Novel interleukin-1 and tumor necrosis factor-alpha modulators, syntheses of said modulators and methods of using said modulators

ABSTRACT

Novel compounds are disclosed that have the chemical structure of Formula (II), and its prodrug esters and acid-addition salts, and that are useful as Interleukin-1 and Tumor Necrosis Factor-α modulators, and thus are useful in the treatment of various listed diseases.  
                 
wherein the R groups are defined as provided in the specification. These compounds are useful, for example, as anti-inflammatory analgesics, in treating immune disorders, as anti-cancer and anti-tumor agents, and in the treatment of cardiovascular disease, skin redness, and viral infection. Completely synthetic and semi-synthetic methods of making these compounds are disclosed, as are methods of using these synthetic and semi-synthetic compounds in the treatment of the above-listed disease states.

PRIORITY CLAIM

The present application is a divisional of U.S. application Ser. No.10/068,333 filed Feb. 4, 2002 which is a continuation of, and claimspriority from U.S. application Ser. No. 09/570,202, filed May 12, 2000(now U.S. Pat. No. 6,365,768), which application claims priority fromU.S. Application Ser. No. 60/134,295, filed May 14, 1999, and U.S.Application Ser. No. 60/186,853, filed Mar. 3, 2000. These applicationsare incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The invention relates to chemical compounds and pharmaceuticalcompositions, including novel chemical compounds and pharmaceuticalcompositions thereof, useful in the treatment of various diseases anddisease states. The invention also relates to methods of synthesizingnatural products and novel, structurally-related chemical compounds.More particularly, the present invention relates to novel analogs of andprocesses for the preparation of compounds and pharmaceuticalcompositions thereof useful in the treatment of, for example,inflammation, cancer, cachexia, cardiovascular disease, diabetes, otitismedia, sinusitis and transplant rejection.

BACKGROUND OF THE INVENTION

Acanthopanax koreanum Nakai (Araliaceae), which is found indigenously inCheju Island, The Republic of Korea, has been used traditionally as aremedy for, for example, neuralgia, paralysis, and lumbago. Varioususeful components, including acanthoic acid, a compound having thechemical structure of Formula (I), have been isolated from the root barkof this tree. Furthermore, certain analogs of the compound of Formula(I), for example, wherein the COOH group is replaced by a methanolicgroup, by a methyl-acetyl ether, by a methyl group, and by amethyl-ester have each also been isolated from the root bark ofAcanthopanax koreanum Nakai (Araliaceae). See Kim, Y. H. and Chung, B.S., J. Nat. Pro., 51, 1080-83 (1988). (The proper chemical names ofthese analogs are provided in this reference.) This reference and allthe other patents and printed publication cited herein are, in theirentirety, incorporated by reference herein.

The compound of Formula (I), also known as acanthoic acid, has beenreported to have certain pharmacological effects, including, forexample, analgesic and anti-inflammatory activity. The compound ofFormula (I) also exhibits very low toxicity; 1000 mg/kg is the minimumlethal dose (MLD) when administered to a rat. See Lee, Y. S.,“Pharmacological Study for (−)-Pimara-9(11), 15-Diene-19-oic Acid, AComponent of Acanthopanax koreanum Nakai,” Doctorate Thesis, Dept. ofPharmacy, Seoul National University, Korea (1990). The compound ofFormula (I) and/or its naturally-occurring analogs, may exhibit theseknown pharmacological effects by inhibiting leukocyte migration andprostaglandin E₂(PGE₂) synthesis, and is a suspected effector of bothInterleukin-1 (IL-1) and Tumor Necrosis Factor-α (TNF-α) production.Additionally, a process for the preparation of acanthoic acid, and useof the acanthoic acid for treatment of immune disease is described inInternational Patent Publication WO 95/34300 (Dec. 21, 1995).

Also, the compound of Formula (IA), kauranoic acid, and thecorresponding methyl-ester analog of the compound of Formula (IA), aswell as methanolic reduction analogs of the compound of Formula (IA)have been isolated from the root bark of Acanthopanax koreanum Nakai(Araliaceae). See Kim, Y. H. and Chung, B. S., J. Nat. Pro., 51, 1080(1988). (The proper chemical name of kauranoic acid,(−)-kaur-16-en-19-oic acid, and of the known analogs of kauranoic acidare provided in this reference.)

Tumor Necrosis Factor-α (herein “TNF-α” or “TNF”) and/or Interleukin-1(herein “IL-1”) are involved in various biochemical pathways and, thusmodulators of TNF-α and/or IL-1 activity or production, especially novelmodulators of TNF-α and/or IL-1 activity or novel compounds thatinfluence the production of either IL-1 or TNF-α, or both, are highlydesired. Such compounds and classes of compounds would be valuable inmaintaining the human immune system and in treating diseases such as forexample, tuberculous pleurisy, rheumatoid pleurisy, and diseases notconventionally considered to be immune disorders, such as cancer,cardiovascular disease, skin redness, viral infection, diabetes, andtransplant rejection.

Although numerous approaches to regulate the production of TumorNecrosis Factor-α and the interleukins are known, novel approaches,compounds, and pharmaceutical formulations to regulate the production ofTumor Necrosis Factor-α and interleukins are highly desirable and havebeen long sought by those of skill in the art.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provideprocesses for the synthetic and semi-synthetic preparation of thecompounds of Formulae (I) and (IA) and their structural analogs,including novel analogs, of the compounds of Formulae (I) and (IA).

The compounds of the present invention include, for example, compoundshaving the chemical structure of Formula (II) and compounds having thechemical structure of Formula (IIA). Regarding compounds having thechemical structure of Formula (II), the invention includes:

-   -   wherein the R groups are defined as follows: If any R₃-R₅, R₇,        R₈, R₁₁-R₁₃ is not hydrogen, R₂ or R₆ or R₉ is not methyl, or        R₁₀ is not CH₂, then R₁ is selected from the group consisting of        hydrogen, a halogen, COOH, C₁-C₁₂ carboxylic acids, C₁-C₁₂ acyl        halides, C₁-C₁₂ acyl residues, C₁-C₁₂ esters, C₁-C₁₂ secondary        amides, (C₁-C₁₂)(C₁-C₁₂) tertiary amides, C₁-C₁₂ alcohols,        (C₁-C₁₂)(C₁-C₁₂) ethers, C₁-C₁₂ alkyls, C₁-C₁₂ substituted        alkyls, C₂-C₁₂ alkenyls, C₂-C₁₂ substituted alkenyls, and C₅-C₁₂        aryls; however, if all R₃-R₅, R₇, R₈, R₁₁-R₁₃ are hydrogen, R₂,        R₆, and R₉ are each methyl, and R₁₀ is CH₂, then R₁ is selected        from hydrogen, a halogen, C₁-C₁₂ carboxylic acids, C₁-C₁₂ acyl        halides, C₁-C₁₂ acyl residues, C₂-C₁₂ esters, C₂-C₁₂ secondary        amides, (C₁-C₁₂)(C₁-C₁₂) tertiary amides, C₂-C₁₂ alcohols,        (C₁-C₁₂)(C₁-C₁₂) ethers other than methyl-acetyl ether, C₂-C₁₂        alkyls, C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyls, C₂-C₁₂        substituted alkenyls, and C₂-C₁₂ aryls.

R₂ and R₉ are each separately selected from hydrogen, a halogen, C₁-C₁₂alkyl, C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substitutedalkenyl, C₂-C₁₂ alkynyl, C₁-C₁₂ acyl, C₁-C₁₂ alcohol, and C₅-C₁₂ aryl.

R₃-R₅, R₇, R₈, and R₁₁-R₁₃ are each separately selected from hydrogen, ahalogen, C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂substituted alkenyl, C₂-C₁₂ alkynyl, and C₅-C₁₂ aryl. In particularlypreferred embodiments, R₁₁ is a C₁-C₆ alkyl, or C₁-C₆ substituted alkyl,and all other R groups are hydrogen.

R₆ is selected from hydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substituted alkenyl, andC₂-C₁₂ alkynyl.

R₁₀ is selected from hydrogen, a halogen, CH₂, C₁-C₆ alkyl, C₁-C₆substituted alkyl, C₂-C₆ alkenyl, C₂-C₆ substituted alkenyl, C₁-C₁₂alcohol, and C₅-C₁₂ aryl.

R₁₄ and R₁₅ are separately selected from hydrogen, a halogen, CH₂, C₁-C₆alkyl, C₁-C₆ substituted alkyl, C₂-C₆ alkenyl, C₂-C₆ substitutedalkenyl, C₁-C₆ alcohol, and C₅-C₆ aryl.

Regarding compounds having the chemical structure of Formula (IIA), theinvention includes:

-   -   wherein, if any R₃-R₅, R₇, R₈, R₁₁-R₁₃ is not hydrogen, R₂ or R₆        is not methyl, R₁₀ is not CH₂, or if it is not true that R₁₀ is        CH₂OH and R₁₁ is OH, then R₁ is selected from the group        consisting of hydrogen, a halogen, COOH, C₁-C₁₂ carboxylic        acids, C₁-C₁₂ acyl halides, C₁-C₁₂ acyl residues, C₁-C₁₂ esters,        C₁-C₁₂ secondary amides, (C₁-C₁₂)(C₁-C₁₂) tertiary amides,        C₁-C₁₂ alcohols, (C₁-C₁₂)(C₁-C₁₂) ethers, C₁-C₁₂ alkyls, C₁-C₁₂        substituted alkyls, C₂-C₁₂ alkenyls, C₂-C₁₂ substituted        alkenyls; but    -   if all R₃-R₅, R₇, R₈, R₁₁-R₁₃ are hydrogen, R₂ and R₆ are each        methyl, and R₁₀ is CH₂ or CH₂OH, then R₁ is selected from        hydrogen, a halogen, C₁-C₁₂ carboxylic acids, C₁-C₁₂ acyl        halides, C₁-C₁₂ acyl residues, C₂-C₁₂ esters, C₁-C₁₂ secondary        amides, (C₁-C₁₂)(C₁-C₁₂) tertiary amides, C₂-C₁₂ alcohols,        (C₁-C₁₂)(C₁-C₁₂) ethers, C₂-C₁₂ alkyls, C₂-C₁₂ substituted        alkyls, C₂-C₁₂ alkenyl, and C₂-C₁₂ substituted alkenyl;    -   R₂ is selected from hydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂        substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substituted alkenyl,        C₂-C₁₂ alkynyl, C₁-C₁₂ acyl, C₁-C₁₂ alcohol, and C₅-C₁₂ aryl;    -   R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ are each separately selected        from hydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂ substituted        alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substituted alkenyl, C₂-C₁₂        alkynyl, and C₅-C₁₂ aryl. In particularly preferred embodiments,        R₁₁ is a C₁-C₆ alkyl, or C₁-C₆ substituted alkyl, and all other        R groups are hydrogen;    -   R₆ is selected from hydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂        substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substituted alkenyl,        and C₂-C₁₂ alkynyl;    -   R₁₀ is selected from hydrogen, a halogen, CH₂, C₁-C₆ alkyl,        C₁-C₆ substituted alkyl, C₂-C₆ alkenyl, C₂-C₆ substituted        alkenyl, C₁-C₁₂ alcohol, and C₅-C₁₂ aryl; and    -   R₁₄ and R₁₅ may be stereo-specific, and are separately selected        from hydrogen, a halogen, CH₂, C₁-C₆ alkyl, C₁-C₆ substituted        alkyl, C₂-C₆ alkenyl, C₂-C₆ substituted alkenyl, C₁-C₆ alcohol,        and C₅-C₆ aryl.

It is a further object of the invention to provide compounds having thechemical structure of Formula (IIB), and to provide processes for thesynthetic and semi-synthetic preparation of compounds having thechemical structure of Formula (IIB). Regarding said compounds having thechemical structure of Formula (IIB), for example, the compounds hereindesignated TTL1, TTL2, TTL3, TTL4, and their analogs and derivatives,the invention includes:

-   -   wherein the R groups are defined as follows: R₁ is selected from        the group consisting of hydrogen, a halogen, COOH, C₁-C₁₂        carboxylic acids, C₁-C₁₂ acyl halides, C₁-C₁₂ acyl residues,        C₁-C₁₂ esters, C₁-C₁₂ secondary amides, (C₁-C₁₂)(C₁-C₁₂)        tertiary amides, C₁-C₁₂ alcohols, (C₁-C₁₂)(C₁-C₁₂) ethers,        C₁-C₁₂ alkyls, C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyls,        C₂-C₁₂ substituted alkenyls, and C₅-C₁₂ aryls. Under these        conditions, R₁ is preferably selected from COOH, C₁-C₁₂        carboxylic acids, C₁-C₁₂ acyl halides, C₁-C₁₂ acyl residues, and        C₁-C₁₂ esters, and is most preferably selected from COOH and the        C₁-C₆ esters.

R₂ and R₉ are each separately selected from hydrogen, a halogen, C₁-C₁₂alkyl, C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substitutedalkenyl, C₂-C₁₂ alkynyl, C₁-C₁₂ acyl, C₁-C₁₂ alcohol, and C₅-C₁₂ aryl.

R₃-R₅, R₇, R₈, and R₁₁-R₁₃ are each separately selected from hydrogen, ahalogen, C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂substituted alkenyl, C₂-C₁₂ alkynyl, and C₅-C₁₂ aryl. In particularlypreferred embodiments, R₁₁ is a C₁-C₆ alkyl, or C₁-C₆ substituted alkyl,and all other R groups are hydrogen.

R₆ is selected from hydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substituted alkenyl, andC₂-C₁₂ alkynyl.

R₁₀ is selected from hydrogen, a halogen, CH₂, C₁-C₆ alkyl, C₁-C₆substituted alkyl, C₂-C₆ alkenyl, C₂-C₆ substituted alkenyl, C₁-C₁₂alcohol, and C₅-C₁₂ aryl.

R₁₄ and R₁₅ are stereo-specific and are separately selected fromhydrogen, a halogen, CH₂, C₁-C₆ alkyl, C₁-C₆ substituted alkyl, C₂-C₆alkenyl, C₂-C₆ substituted alkenyl, C₁-C₆ alcohol, and C₅-C₆ aryl.

It also will be appreciated that the various R groups, most particularlyR₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃, may be chosen such that cyclic systemare formed. For example, both R₁₃ and R₁₂ may be ethylene moieties andmay include a covalent C—C linkage between their respective terminalcarbons, generating an additional six-membered ring in the compound ofFormula (IIB). As a further example, bis-cyclic rings may be formed bychoosing appropriate chemical species for the various R groups, mostparticularly R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ of Formula (IIB).

The compounds of the invention include the prodrug esters of thecompounds of Formulae (II), (IIA), and (IIB), and the acid-additionsalts of the compounds of Formulae (II), (IIA), and (IIB), andpharmaceutical compositions comprising a therapeutically effectiveamount of the described compounds, including their prodrug esters andtheir acid-addition salts, optionally in conjunction with apharmaceutically acceptable carrier. Such compositions are useful as,for example, anti-inflammatory analgesics, in the treatment of immuneand auto-immune disorders, as anti-cancer or anti-tumor agents, and areuseful in the treatment of cardiovascular disease, skin redness, viralinfection, diabetes, otitis media, sinusitis and/or transplantrejection. Particularly, a pharmaceutical composition comprising atherapeutically effective amount of a compound of Formulae (II), (IIA),or (IIB), or a pro-drug ester and acid addition salt of a compound ofFormulae (II), (IIA), or (IIB), may be used as an anti-cancer,anti-tumor agent, anti-viral agent, and may be useful in the treatmentof cardiovascular disease, skin redness, viral infection, diabetes,otitis media, sinusitis and/or transplant rejection.

The invention also provides novel methods of synthesizing the abovedescribed compounds and their analogs comprising the step of performinga Diels-Alder reaction reacting a diene having two or more rings with adienophile compound to yield a resultant compound have three of morerings; and yielding a desired synthetic compound. The Diels-Alderreaction, along with the selection of the diene and the dienophileaffords flexibility in synthesizing a variety of compounds of theinvention, and allows for the use of combinatorial chemistry librariesof compounds of the invention, for use biological assays, includingclinical trials.

BRIEF DESCRIPTION OF THE FIGURES

Certain preferred embodiments of the invention are illustrated in theFigures. The Figures merely illustrate certain preferred embodiments ofthe invention and/or certain preferred methods of making and/or of usingthe invention. The Figures are not intended to limit the scope of theinvention described and claimed herein.

FIG. 1 depicts the structure of acanthoic acid and acanthoic acid methylester, a stereo chemical view of acanthoic acid, and a skeletal-typeview of certain compounds of the invention.

FIG. 2 depicts the retrosynthetic analysis and strategic bondassociations of certain compounds of the invention.

FIG. 3 depicts selected approaches to the construction of the AB ring ofcertain compounds of the invention including: Wenkert's approach to thesynthesis of (±) podocapric acid; Welch's approach to the synthesis of(±) podocapric acid; and DeGrot's approach to the synthesis of (±)podocapric acid.

FIG. 4 depicts a schematic synthetic scheme (Scheme 1) of the synthesisof the AB ring system of acanthoic acid and certain compounds of theinvention.

FIG. 5 depicts a synthetic scheme (Scheme 2) by which the synthesis ofacanthoic acid and certain compounds of the invention may be completed.

FIG. 6 depicts the minimized, three-dimensional model of diene 42, asdescribed in the detailed description of the invention.

FIG. 7 depicts a synthetic scheme (Scheme 3) for the development andapplication of catalyst 49, as described in the detailed description ofthe preferred embodiment of the invention, an asymmetric Diels-Alderreaction.

FIG. 8 depicts a synthetic scheme (Scheme 4) for the synthesis of thecompound of Formula (I) and certain compounds of the invention based onan asymmetric Diels-Alder methodology.

FIG. 9 depicts the structure activity relationship and the focus ofstructure activity relationship studies of oleanolic acid and itsderivatives and certain compounds of the invention.

FIG. 10 depicts sites identified for the structural alteration andstructure activity relationship studies of Compound 1.

FIG. 11 depicts preferred, representative examples of analogs ofCompound 1 for use in structure activity relationship studies andchemical biological studies.

FIG. 12 depicts certain preferred, representative derivatives ofCompound 1 for photo affinity labeling studies.

FIG. 13 depicts certain preferred, representative examples of dimersand/or conjugates of Compound 1.

FIG. 14 depicts a complete chemical synthesis of certain compounds ofthe invention, identified herein as TTL1 and TTL3 in FIG. 17.

FIG. 15 depicts a chemical synthesis of a preferred ¹⁴C-labeled compoundof the invention, identified as TTL3 in FIG. 17.

FIG. 16 depicts the complete chemical synthesis of the compound ofFormula (I).

FIG. 17 depicts a summary of the syntheses of certain compounds of theinvention, and the physical properties of these compounds. CompoundsTTL1, TTl2, TTL3, and TTL4 are defined as depicted in this Figure.

FIG. 18 depicts a summary of the synthesis of Example 1.

FIG. 19 depicts the structures of (−) acanthoic acid and (+) pimaricacid.

FIG. 20 depicts the retrosynthetic analysis of (−) acanthoic acid ofExample 1.

FIG. 21 depicts the synthetic scheme (Scheme 5) of preferred compoundsof Formula (IIB) as described in Examples 1-6. The regents, conditions,and percentage yields of each step were as follows: (a) 0.1 equiv PTSA(CH₂OH)₂, benzene, 80° C., 4 h, 90%; (b) 2.2 equiv Li, liquid NH₃, 1.0equiv tBuOH, −78 to −30° C., 30 minu then isoprene (excess), −78 to 50°C.; 1.1 equiv NC—CO₂Me, Et₂O, −78 to 0° C., 2 h, 55%; (c) 1.1 equiv NaH,HMPA, 25° C., 3 h; 1.,1 equiv MoMCI, 25° C., 2 h, 95%; (d) 7.0 equiv Li,liquid NH₃, −78 to −30° C., 20 min; CH₃I (excess), −78 to −30° C., 1 h,61%; (e) 1N HCl, THF, 25° C., 15 min, 95%; (f) 1.6 equiv Li acetylide,Et₂O, 25° C., 1 h, 91%; (g) Lindlar's catalyst (20% per weight), H₂,dioxane/pyridine 10/1. 25°, 10 min 95%; (h) 4.4 equiv BF₃.Et₂O,benzene/THF4/1. 80° C., 5 h, 95%; (I) 13 equiv compound 103, neat, 8 h,25° C., 100%; (j) 1.4 equiv NaBH₄, THF MeOH: 10/1, 30 min, 25° C., 94%;(k) 1.1 equiv p-Br—C₆H₄COCl, 1.5 equiv pyridine, 0.1 equiv DMAP, CH₂Cl₂,25°, 2 h, 95% for compound 116, 97% for compound 117.

FIG. 22 depicts the Chem3D representation of ORTEP drawings of compound116 and 117, showing only selected hydrogen atoms for sake of clarity.

FIG. 23 depicts the synthetic scheme (Scheme 6) of the tricyclic core of(−) acanthoic acid of Example 1. The reagents, conditions, andpercentage yields of each step were as follows: (a) 3.0 equiv PhSH, 0.05equiv AIBN, xylenes, 120° C., 18 h, 86%, (b) 1.1 equiv. POCl₃, HMPA, 25°C., 1 h; 1.1 equiv pyridine, 150° C., 18 h 81%; (c) 3.0 equiv compound103, 0.2 equiv SnCl₄ (1 M in CH₂Cl₂), CH₂Cl₂, −20 to 0° C., 20 h, 84%;(d) 1.4 equiv NaBH₄, EtOH, 25° C., 30 min; (e) RaneyNi (excess), THF,65° C., 10 min 91% (over two steps); (f) 1.3 equiv Dess-Martinperiodinane, CH₂Cl₂, 25° C., 30 min; (g) 2.7 equiv P₃PhCH₃Br, 2.2. equivNaHMDS (1.0 in THF), THF, 25° C., 18 h, 86% (over two steps); (h) 3.0LiBr, DMF, 160° C., 3 h, 93%.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Certain compounds of the invention have the chemical structure shown inFormula (II).

The R-groups of the compound of Formula (II) may be selected in thefollowing manner. In the event that (1) any R₃-R₅, R₇, R₈, R₁₁-R₁₃ isnot hydrogen, (2) R₂, R₆ or R₉ is not methyl, or (3) R₁₀ is not CH₂, R₁is selected from the group consisting of hydrogen, a halogen, COOH,C₁-C₁₂ carboxylic acids, C₁-C₁₂ acyl halides, C₁-C₁₂ acyl residues,C₁-C₁₂ esters, C₁-C₁₂ secondary amides, (C₁-C₁₂)(C₁-C₁₂) tertiaryamides, C₁-C₁₂ alcohols, (C₁-C₁₂)(C₁-C₁₂) ethers, C₁-C₁₂ alkyls, C₁-C₁₂substituted alkyls, C₂-C₁₂ alkenyls, C₂-C₁₂ substituted alkenyls, andC₅-C₁₂ aryls. Under these conditions, R₁ is preferably selected fromCOOH, C₁-C₁₂ carboxylic acids, C₁-C₁₂ acyl halides, C₁-C₁₂ acylresidues, and C₁-C₁₂ esters, and is most preferably selected from COOHand the C₁-C₆ esters.

However, in the event that (1) all R₃-R₅, R₇, R₈, R₁₁-R₁₃ are hydrogen,(2) R₂, R₆, and R₉ are each methyl, and (3) R₁₀ is CH₂, R₁ is selectedfrom R₁ is selected from hydrogen, a halogen, C₁-C₁₂ carboxylic acids,C₁-C₁₂ acyl halides, C₁-C₁₂ acyl residues, C₂-C₁₂ esters, C₂-C₁₂secondary amides, (C₁-C₁₂)(C₁-C₁₂) tertiary amides, C₂-C₁₂ alcohols,(C₁-C₁₂)(C₁-C₁₂) ethers other than methyl-acetyl ether, C₂-C₁₂ alkyls,C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyls, C₂-C₁₂ substituted alkenyls,and C₂-C₁₂ aryls. Under these conditions, R₁ is preferably selected fromC₁-C₁₂ carboxylic acids, C₁-C₁₂ acyl halides, C₁-C₁₂ acyl residues, andC₂-C₁₂ esters, and is most preferably a C₄-C₈ ester.

R₂ and R₉ are each separately selected from hydrogen, a halogen, C₁-C₁₂alkyl, C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substitutedalkenyl, C₂-C₁₂ alkynyl, C₁-C₁₂ acyl, C₁-C₁₂ alcohol, and C₅-C₁₂ aryl.Preferably, R₂ and R₉ are each separately selected from the alkyl andalkenyl residues. Most preferably, R₂ and R₉ are each methyl residues,although one of R₂ and R₉ may be methyl and the other not methyl inpreferred embodiments of the compound of Formula (II).

R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ are each separately selected fromhydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyls, C₂-C₁₂alkenyl, C₂-C₁₂ substituted alkenyl, C₂-C₁₂ alkynyl, and C₅-C₁₂ aryl.Preferably, R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ are each hydrogen or a C₁-C₆alkyl, and most preferably R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ are eachhydrogen. Nevertheless, any one or several of R₃, R₄, R₅, R₇, R₈, andR₁₁-R₁₃ may be hydrogen, while the others may be not hydrogen, inpreferred embodiments of the compound of Formula (II).

R₆ is selected from hydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substituted alkenyl, andC₂-C₁₂ alkynyl. Preferably, R₆ is selected from hydrogen, a halogen,C₁-C₆ alkyl. More preferably, R₆ is a C₁-C₆ alkyl, and most preferably,R₆ is methyl.

R₁₀ is selected from hydrogen, a halogen, CH₂, C₁-C₆ alkyl, C₁-C₆substituted alkyl, C₂-C₆ alkenyl, C₂-C₆ substituted alkenyl, C₁-C₁₂alcohol, and C₅-C₁₂ aryl. The bond linking R₁₀ to the remainder of thecompound of Formula (II) is preferably a C—C double bond, but may be aC—C single bond, a C—H single bond, or a heteroatomic single bond.Preferably, R₁₀ is CH₂ or CH₂R′ wherein R′ is a C₁-C₆ alkyl, or a C₁-C₆substituted alkyl. Most preferably, R₁₀ is CH₂.

R₁₄ and R₁₀ are separately selected from hydrogen, a halogen, CH₂, C₁-C₆alkyl, C₁-C₆ substituted alkyl, C₂-C₆ alkenyl, C₂-C₆ substitutedalkenyl, C₁-C₆ alcohol, and C₅-C₆ aryl, with hydrogen and C₁-C₆ alkyl,C₁-C₆ substituted alkyl most preferred.

It also will be appreciated that the various R groups, most particularlyR₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃, may be chosen such that cyclic systemare formed. For example, both R₁₃ and R₁₂ may be ethylene moieties andmay include a covalent C—C linkage between their respective terminalcarbons, generating an additional six-membered ring in the compound ofFormula (II). As a further example, bis-cyclic rings may be formed bychoosing appropriate chemical species for the various R groups, mostparticularly R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃,of Formula (II).

Certain preferred compounds of the present invention have the structureshown in Formula (IIA).

The R-groups of the compound of Formula (IIA) may be selected in thefollowing manner. In the event that if any R₃-R₅, R₇, R₈, R₁₁-R₁₃ is nothydrogen, R₂ or R₆ is not methyl, R₁₀ is not CH₂, or if it is not truethat R₁₀ is CH₂OH and R₁₁ is OH, R₁ is selected from the groupconsisting of hydrogen, a halogen, COOH, C₁-C₁₂ carboxylic acids, C₁-C₁₂acyl halides, C₁-C₁₂ acyl residues, C₁-C₁₂ esters, C₁-C₁₂ secondaryamides, (C₁-C₁₂)(C₁-C₁₂) tertiary amides, C₁-C₁₂ alcohols,(C₁-C₁₂)(C₁-C₁₂) ethers, C₁-C₁₂ alkyls, C₁-C₁₂ substituted alkyls,C₂-C₁₂ alkenyls, C₂-C₁₂ substituted alkenyls. Under these conditions, R₁is preferably selected from COOH, C₁-C₁₂ carboxylic acids, C₁-C₁₂ acylhalides, C₁-C₁₂ acyl residues, and C₁-C₁₂ esters, and is most preferablyselected from COOH and the C₁-C₆ esters.

In the event that all R₃-R₅, R₇, R₈, R₁₁-R₁₃ are hydrogen, R₂ and R₆ areeach methyl, and R₁₀ is CH₂ or CH₂OH, R₁ is selected from hydrogen, ahalogen, C₁-C₁₂ carboxylic acids, C₁-C₁₂ acyl halides, C₁-C₁₂ acylresidues, C₂-C₁₂ esters, C₁-C₁₂ secondary amides, (C₁-C₁₂)(C₁-C₁₂)tertiary amides, C₂-C₁₂ alcohol, (C₁-C₁₂)(C₁-C₁₂) ethers, C₂-C₁₂ alkyls,C₂-C₁₂ substituted alkyls, C₂-C₁₂ alkenyl, and C₂-C₁₂ substitutedalkenyl. Under these conditions, R₁ is preferably selected from C₁-C₁₂carboxylic acids, C₁-C₁₂ acyl halides, C₁-C₁₂ acyl residues, and C₂-C₁₂esters, and is most preferably a C₄-C₈ ester.

R₂ is selected from hydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substituted alkenyl, C₂-C₁₂alkynyl, C₁-C₁₂ acyl, C₁-C₁₂ alcohol, and C₅-C₁₂ aryl. Preferably, R₂and R₉ are each separately selected from the alkyl and alkenyl residues.Most preferably, R₂ and R₉ are each methyl residues, although one of R₂and R₉ may be methyl and the other not methyl in preferred embodimentsof the compound of Formula (IIA).

R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ are each separately selected fromhydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyls, C₂-C₁₂alkenyl, C₂-C₁₂ substituted alkenyl, C₂-C₁₂ alkynyl, and C₅-C₁₂ aryl.Preferably, R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ are each hydrogen or a C₁-C₆alkyl, and most preferably R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ are eachhydrogen. Nevertheless, any one or several of R₃, R₄, R₅, R₇, R₈, andR₁₁-R₁₃ may be hydrogen, while the others may be not hydrogen, inpreferred embodiments of the compound of Formula (IIA).

R₆ is selected from hydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substituted alkenyl, andC₂-C₁₂ alkynyl. Preferably, R₆ is selected from hydrogen, a halogen,C₁-C₆ alkyl. More preferably, R₆ is a C₁-C₆ alkyl, and most preferably,R₆ is methyl.

R₁₀ is selected from hydrogen, a halogen, CH₂, C₁-C₆ alkyl, C₁-C₆substituted alkyl, C₂-C₆ alkenyl, C₂-C₆ substituted alkenyl, C₁-C₁₂alcohol, and C₅-C₁₂ aryl. The bond linking R₁₀ to the remainder of thecompound of Formula (IIA) is preferably a C—C double bond, but may be aC—C single bond, a C—H single bond, or a heteroatomic single bond.Preferably, R₁₀ is CH₂ or CH₂R′ wherein R′ is a C₁-C₆ alkyl, or a C₁-C₆substituted alkyl. Most preferably, R₁₀ is CH₂.

It also will be appreciated that the various R groups, most particularlyR₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃, may be chosen such that cyclic systemare formed. For example, both R₁₃ and R₁₂ may be ethylene moieties andmay include a covalent C—C linkage between their respective terminalcarbons, generating an additional six-membered ring in the compound ofFormula (IIA). As a further example, bis-cyclic rings may be formed bychoosing appropriate chemical species for the various R groups, mostparticularly R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ of Formula (IIA).

Certain preferred compounds of the present invention, includingcompounds herein designated TTL1, TTL2, TTL3, and TTL4 have the chemicalstructure described in Formula (IIB).

The R-groups of the compound of Formula (IIB) may be selected in thefollowing manner: R₁ is selected from the group consisting of hydrogen,a halogen, COOH, C₁-C₁₂ carboxylic acids, C₁-C₁₂ acyl halides, C₁-C₁₂acyl residues, C₁-C₁₂ esters, C₁-C₁₂ secondary amides, (C₁-C₁₂)(C₁-C₁₂)tertiary amides, C₁-C₁₂ alcohols, (C₁-C₁₂)(C₁-C₁₂) ethers, C₁-C₁₂alkyls, C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyls, C₂-C₁₂ substitutedalkenyls, and C₅-C₁₂ aryls. Under these conditions, R₁ is preferablyselected from COOH, C₁-C₁₂ carboxylic acids, C₁-C₁₂ acyl halides, C₁-C₁₂acyl residues, and C₁-C₁₂ esters, and is most preferably selected fromCOOH and the C₁-C₆ esters.

R₂ and R₉ are each separately selected from hydrogen, a halogen, C₁-C₁₂alkyl, C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substitutedalkenyl, C₂-C₁₂ alkynyl, C₁-C₁₂ acyl, C₁-C₁₂ alcohol, and C₅-C₁₂ aryl.Preferably, R₂ and R₉ are each separately selected from the alkyl andalkenyl residues. Most preferably, R₂ and R₉ are each methyl residues,although one of R₂ and R₉ may be methyl and the other not methyl inpreferred embodiments of the compound of Formula (IIB).

R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ are each separately selected fromhydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyls, C₂-C₁₂alkenyl, C₂-C₁₂ substituted alkenyl, C₂-C₁₂ alkynyl, and C₅-C₁₂ aryl.Preferably, R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ are each hydrogen or a C₁-C₆alkyl, and most preferably R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ are eachhydrogen. Nevertheless, any one or several of R₃, R₄, R₅, R₇, R₈, andR₁₁, —R₁₃ may be hydrogen, while the others may be not hydrogen, inpreferred embodiments of the compound of Formula (IIB).

R₆ is selected from hydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substituted alkenyl, andC₂-C₁₂ alkynyl. Preferably, R₆ is selected from hydrogen, a halogen,C₁-C₆ alkyl. More preferably, R₆ is a C₁-C₆ alkyl, and most preferably,R₆ is methyl.

R₁₀ is selected from hydrogen, a halogen, CH₂, C₁-C₆ alkyl, C₁-C₆substituted alkyl, C₂-C₆ alkenyl, C₂-C₆ substituted alkenyl, C₁-C₁₂alcohol, and C₅-C₁₂ aryl. The bond linking R₁₀ to the remainder of thecompound of Formula (II) is preferably a C—C double bond, but may be aC—C single bond, a C—H single bond, or a heteroatomic single bond.Preferably, R₁₀ is CH₂ or CH₂R′ wherein R′ is a C₁-C₆ alkyl, or a C₁-C₆substituted alkyl. Most preferably, R₁₀ is CH₂.

It also will be appreciated that the various R groups, most particularlyR₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃, may be chosen such that cyclic systemare formed. For example, both R₁₃ and R₁₂ may be ethylene moieties andmay include a covalent C—C linkage between their respective terminalcarbons, generating an additional six-membered ring in the compound ofFormula (IIB). As a further example, bis-cyclic rings may be formed bychoosing appropriate chemical species for the various R groups, mostparticularly R₃, R₄, R₅, R₇, R₈, and R₁₁-R₁₃ of Formula (IIB).

Definitions

As used herein, the term “alkyl” means any unbranched or branched,saturated hydrocarbon, with C₁-C₆ unbranched, saturated, unsubstitutedhydrocarbons being preferred, and with methyl, ethyl, iosbutyl, andtert-butyl being most preferred. Among the substituted, saturatedhydrocarbons, C₁-C₆ mono- and di- and pre-halogen substituted saturatedhydrocarbons and amino-substituted hydrocarbons are preferred, withperfluromethyl, perchloromethyl, perfluoro-tert-butyl, andperchloro-tert-butyl being the most preferred. The term “substitutedalkyl” means any unbranched or branched, substituted saturatedhydrocarbon, with unbranched C₁-C₆ alkyl secondary amines, substitutedC₁-C₆ secondary alkyl amines, and unbranched C₁-C₆ alkyl tertiary aminesbeing within the definition of “substituted alkyl,” but not preferred.The term “substituted alkyl” means any unbranched or branched,substituted saturated hydrocarbon. Cyclic compounds, both cyclichydrocarbons and cyclic compounds having heteroatoms, are within themeaning of “alkyl.”

As used herein, the term “substituted” means any substitution of ahydrogen atom with a functional group.

As used herein, the term “functional group” has its common definition,and refers to chemical moieties preferably selected from the groupconsisting of a halogen atom, C₁-C₂₀ alkyl, substituted C₁-C₂₀ alkyl,perhalogenated alkyl, cyloalkyl, substituted cycloalkyl, aryl,substituted aryl, benzyl, heteroaryl, substituted heteroaryl, cyano, andnitro. Functional groups may also be selected from the group consistingof —SR_(S), —OR_(O), —NR_(n1)R_(n2), —N⁺R_(n1)R_(n2)R_(n3), —N═N—R_(n1),—P⁺R_(n1)R_(n2)R_(n3), —COR_(C), —C(═NOR_(O))R_(C), —CSR_(C), —OCOR_(C),—OCONR_(n1)R_(n2), —OCO₂R_(C), —CONR_(n1)R_(n2), —C(═NOR)NR_(n1)R_(n2),—CO₂R_(O), —SO₂NR_(n1)R_(n2), —SO₃R_(O), —SO₂R_(O), —PO(OR_(O))₂,—NR_(n1)CSNR_(n2)R_(n3). Substituents of these functional groups R_(n1),R_(n2), R_(n3), R_(O) and R_(S) are preferably each separately selectedfrom the group consisting of a hydrogen atom, C₁-C₂₀ alkyl, substitutedC₁-C₂₀ alkyl, cyloalkyl, substituted cycloalkyl, aryl, substituted aryl,benzyl, heteroaryl, substituted heteroaryl and may constitute parts ofan aliphatic or aromatic heterocycle. R_(C) are preferably selected fromthe group consisting of a hydrogen atom, C₁-C₂₀ alkyl, substitutedC₁-C₂₀ alkyl, perhalogenated alkyl, cyloalkyl, substituted cycloalkyl,aryl, substituted aryl, benzyl, heteroaryl, substituted heteroaryl andcyano.

As used herein, the terms “halogen” and “halogen atom” refer to any oneof the radio-stable atoms of column 17 of the Periodic Table of theElements, preferably fluorine, chlorine, bromine, or iodine, withfluorine and chlorine being particularly preferred.

As used herein, the term “alkenyl” means any unbranched or branched,substituted or unsubstituted, unsaturated hydrocarbon, with C₁-C₆unbranched, mono-unsaturated and di-unsaturated, unsubstitutedhydrocarbons being preferred, and mono-unsaturated, di-halogensubstituted hydrocarbons being most preferred. The term “substitutedalkenyl” means any unbranched or branched, substituted unsaturatedhydrocarbon, substituted with one or more functional groups, withunbranched C₂-C₆ alkenyl secondary amines, substituted C₂-C₆ secondaryalkenyl amines, and unbranched C₂-C₆ alkenyl tertiary amines beingwithin the definition of “substituted alkyl.” The term “substitutedalkenyl” means any unbranched or branched, substituted unsaturatedhydrocarbon. Cyclic compounds, both unsaturated cyclic hydrocarbons andcyclic compounds having heteroatoms, are within the meaning of“alkenyl.”

As used herein, the term “alcohol” means any unbranched or branchedsaturated or unsaturated alcohol, with C₁-C₆ unbranched, saturated,unsubstituted alcohols being preferred, and with methyl, ethyl,isobutyl, and tert-butyl alcohol being most preferred. Among thesubstituted, saturated alcohols, C₁—C₆ mono- and di-substitutedsaturated alcohols are preferred. The term “alcohol” includessubstituted alkyl alcohols, and substituted alkenyl alcohols.

As used herein, the term “aryl” encompasses the terms “substitutedaryl,” “heteroaryl,” and “substituted heteroaryl” which refer toaromatic hydrocarbon rings, preferably having five or six atomscomprising the ring. The terms “heteroaryl” and “substituted heteroaryl”refer to aromatic hydrocarbon rings in which at least one heteroatom,for example, oxygen, sulfur, or nitrogen atom, is in the ring along withat least one carbon atom. “Aryl,” most generally, and “substitutedaryl,” “heteroaryl,” and “substituted heteroaryl” more particularly,refer to aromatic hydrocarbon rings, preferably having five or sixatoms, and most preferably having six atoms comprising the ring. Theterm “substituted aryl” includes mono and poly-substituted aryls,substituted with, for example, alkyl, aryl, alkoxy, azide, amine, andamino groups. “Heteroaryl” and “substituted heteroaryl,” if usedseparately, specifically refer to aromatic hydrocarbon rings in which atleast one heteroatom, for example, oxygen, sulfur, or nitrogen atom, isin the ring along with at least one carbon atom.

The terms “ether” and “alkoxy” refer to any unbranched, or branched,substituted or unsubstituted, saturated or unsaturated ether, with CI—C₆ unbranched, saturated, unsubstituted ethers being preferred, withdimethyl, diethyl, methyl-isobutyl, and methyl-tert-butyl ethers beingmost preferred. The terms “ether” and “alkoxy,” most generally, and“cycloalkoxy” and cyclic ether” more particularly, refer to anynon-aromatic hydrocarbon ring, preferably having five to twelve atomscomprising the ring.

The term “ester” refer to any unbranched, or branched, substituted orunsubstituted, saturated or unsaturated ester, with C₁-C₆ unbranched,saturated, unsubstituted esters being preferred, with methyl ester, andisobutyl ester being most preferred.

The term “pro-drug ester,” especially when referring to a pro-drug esterof the compound of Formula (I), refers to a chemical derivative of thecompound that is rapidly transformed in vivo to yield the compound, forexample, by hydrolysis in blood. The term “pro-drug ester” refers toderivatives of the compound of the present invention formed by theaddition of any of several ester-forming groups that are hydrolyzedunder physiological conditions. Examples of pro-drug ester groupsinclude pivoyloxymethyl, acetoxymethyl, phthalidyl, indanyl andmethoxymethyl, as well as other such groups known in the art, includinga (5-R-2-oxo-1,3-dioxolen-4-yl)methyl group. Other examples of pro-drugester groups can be found in, for example, T. Higuchi and V. Stella, in“Pro-drugs as Novel Delivery Systems”, Vol. 14, A.C.S. Symposium Series,American Chemical Society (1975); and “Bioreversible Carriers in DrugDesign: Theory and Application”, edited by E. B. Roche, Pergamon Press:New York, 14-21 (1987) (providing examples of esters useful as prodrugsfor compounds containing carboxyl groups).

The term “pharmaceutically acceptable salt,” especially when referringto a pharmaceutically acceptable salt of the compound of Formula (I),refers to any pharmaceutically acceptable salts of a compound, andpreferably refers to an acid addition salt of a compound. Preferredexamples of pharmaceutically acceptable salt are the alkali metal salts(sodium or potassium), the alkaline earth metal salts (calcium ormagnesium), or ammonium salts derived from ammonia or frompharmaceutically acceptable organic amines, for example C₁-C₇alkylamine, cyclohexylamine, triethanolamine, ethylenediamine ortris-(hydroxymethyl)-aminomethane. With respect to compounds of theinvention that are basic amines, the preferred examples ofpharmaceutically acceptable salts are acid addition salts ofpharmaceutically acceptable inorganic or organic acids, for example,hydrohalic, sulfuric, phosphoric acid or aliphatic or aromaticcarboxylic or sulfonic acid, for example acetic, succinic, lactic,malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic,p-toluensulfonic or naphthalenesulfonic acid. Preferred pharmaceuticalcompositions of the present invention include pharmaceuticallyacceptable salts and pro-drug esters of the compound of Formulae (II),(IIA), and (IIB).

The terms “purified,” “substantially purified,” and “isolated” as usedherein refer to the compound of the invention being free of other,dissimilar compounds with which the compound of the invention isnormally associated in its natural state, so that the compound of theinvention comprises at least 0.5%, 1%, 5%, 10%, or 20%, and mostpreferably at least 50% or 75% of the mass, by weight, of a givensample. In one preferred embodiment, these terms refer to the compoundof the invention comprising at least 95% of the mass, by weight, of agiven sample.

The terms “anti-cancer,” “anti-tumor” and “tumor-growth-inhibiting,”when modifying the term “compound,” and the terms “inhibiting” and“reducing”, when modifying the terms “compound” and/or the term “tumor,”mean that the presence of the subject compound is correlated with atleast the slowing of the rate of growth of the tumor or cancerous mass.More preferably, the terms “anti-cancer,” “anti-tumor,”“tumor-growth-inhibiting,” “inhibiting,” and “reducing” refer to acorrelation between the presence of the subject compound and at leastthe temporary cessation of tumor growth or growth of the cancerous mass.The terms “anti-cancer,” “anti-tumor,” “tumor-growth-inhibiting,”“inhibiting,” and “reducing” also refer to, particularly in the mostpreferred embodiment of the invention, a correlation between thepresence of the subject compound and at least the temporary reduction inthe mass of the tumor. These terms refer to cancer and variousmalignancies in animals, specifically in mammals, and most specificallyin humans.

The term “skin redness” means any skin redness, especially a chronicskin redness having a neurogenic origin, consistent with, but notlimited by, its meaning in EP 7744250, which is hereby incorporated byreference herein in its entirety.

The term “viral infection” means any infection of a viral originincluding rhinovirus, and preferably, but not exclusively, refers tohuman immunodeficiency virus (HIV), human cytomegalovirus, hepatitis A,hepatitis B, and hepatitis C viruses.

The term “cardiovascular disease” refers to the various diseases of theheart and vascular systems, including but not limited to congestiveheart failure, cardiac dysfunction, reperfusion injury, and variousknown peripheral circulatory abnormalities. “Cardiovascular disease”refers to such diseases in animals, specifically in mammals, and mostspecifically in humans.

As used herein, the term “diabetes” refers to the various diseasesrelated to elevated insulin levels, Insulin Resistance, or Diabetes,including Type 1 Diabetes, Type 2 Diabetes, and various relatedcondition, including, but not limited to Stein-Leventhal Syndrome orPolycystic Ovary Syndrome (PCOS).

As used herein, the term “transplant rejection” refers to theconditions, and related symptoms known as allograft rejection, xenograftrejection, and autograft rejection, and in preferred embodiments of theinvention, refers to human-human allograft rejection.

As used herein, the terms “modulator” or “modulation” refer to thecapacity of a compound or course of treatment to alter the presence orproduction of a modulated compound, especially TNF-α or IL-1, in anindividual. Most preferably, “modulator” or “modulation” refer to thecapacity of a compound or course of treatment to reduce the presence orproduction of a modulated compound.

As used herein, the terms TTL1, TTL2, TTL3, TTL4 and TTL5 refer to thespecific chemical entities identified in, among other figures, FIG. 17.

All other chemical, medical, pharmacological, or otherwise technicalterms used herein are to be understood as they would be understood bypersons of ordinary skill in the art.

Interleukin-1 (IL-1)

Interleukin-1 (IL-1) is a regulatory factor which participates in a widerange of mammalian immune and inflammatory mechanisms and otherdefensive mechanism, especially mechanisms in the human body. See, e.g.,Dinarello, D. A., FASEB J., 2, 108 (1988). IL-1, first discovered asproduced by activated macrophages, is secreted by various cells, forexample, fibroblasts, keratinocytes, T cells, B cells, and astrocytes ofthe brain, and has been reported to have various functions including:stimulating the proliferation of CD4+ T cells, see Mizel, S. B.,Immunol. Rev., 63, 51 (1982); stimulating the cell-killing effect ofthymic T_(C) cells through its binding to a T cell receptor, TCR, seeMcConkey, D. J., et al., J. Biol. Chem., 265, 3009(1990); inducing theproduction of various materials participating in the inflammatorymechanisms, for example, PGE₂, phospholipase A₂ (PLA₂) and collagenase,see Dejana, E., et al., Bolid, 69, 695-699 (1987)); inducing theproduction of acute-phase proteins in liver, see Andus, T., et al., Eur.J. Immunol., 123, 2928 (1988)); raising blood pressure in the vascularsystem, see Okusawa, S., et al., J. Clin. Invest., 81, 1162 (1988)); andinducing the production of other cytokines, for example, IL-6 and TNF-α,see Dinarello, C. A., et al., J. Immunol., 139, 1902(1987). IL-1modulation is also known to effect rheumatoid arthritis, see Nouri, A.M., et al., Clin. Exp. Immunol., 58, 402(1984); transplant rejection,see Mauri and Teppo, Transplantation, 45, 143 (1988); and septicemia,see Cannon, J. G., et al., Lymphokine Res., 7, 457 (1988), and IL-1 mayinduce fever and pain when administered in large doses. See Smith, J.,et al., Am. Soc. Clin. Oncol., 9, 710 (1990)).

The occurrence of septicemia, arthritis, inflammations, and relatedconditions in animal models can be decreased by inhibiting IL-1 bindingto its receptors by employing naturally occurring IL-1 receptorinhibitors (IL-1 Ra), see Dinarello, C. A. and Thompson, R. C., Immunol.Today, 12, 404 (1991), and certain methods for inhibiting the activityof IL-1 by employing particular antibodies have been proposed, seeGiovine, D. F. S. and Duff, G. W., Immunol. Today. 11, 13 (1990). Incase of IL-6, proliferation of myelocytes in a patient suffering frommyeloma which is caused by an excessive secretion of IL-6 has beensuppressed by employing antibodies against IL-6 or IL-6 receptor, seeSuzuki, H., Eur. J. Immuno., 22, 1989(1992)). The disease conditiontreatable according to the invention, via TNF-α and IL-1 modulationinduced by the compounds of the invention, include but are notnecessarily limited to the disease conditions herein described.

Tumor Necrosis Factor-α (TNF-α)

Human TNF-α was first purified in 1985. See Aggarwal, B. B.; Kohr, W. J.“Human tumor necrosis factor. Production, purification andcharacterization”. J. Biol. Chem. 1985, 260, 2345-2354. Soon after, themolecular cloning of the TNF cDNA and the cloning of the human TNF locuswere accomplished. See Pennica, D.; Nedwin, G. E.; Hayflick, J. S. et al“Human necrosis factor: precursor structure, expression and homology tolymphotoxin”. Nature 1984, 312, 724-729. Wang, A. M.; Creasy, A. A.;Ladner, M. B. “Molecular cloning of the complementary DNA for humanTumor Necrosis Factor”. Nature 1985, 313, 803-806. TNF-α is a trimeric17-KDa polypeptide mainly produced by macrophages. This peptide isinitially expressed as a 26-KDa transmembrane protein from which the17-KDa subunit is cleaved and released following proteolytic cleavage byan enzyme known as TACE. This work clarified the immense andmultifaceted biological implications of TNF-α and spurred thedevelopment of therapeutic approaches targeting its overproduction.

Tumor necrosis Factor-α (TNF-α), is typically produced by various cells,for example, activated macrophages and fibroblasts. TNF-α has beenreported to induce IL-1 production, see Dinarello, D. A., FASEB J., 2,108 (1988), kill the fibrosarcoma L929 cells, see Espevik andNissen-Meyer, J. Immunol. Methods, 95, 99 (1986); to stimulate theproliferation of fibroblasts, see Sugarman, B. J., et al., Science, 230,943(1985); to induce the production of PGE₂ and arachidonic acid, bothof which may be involved in inflammatory responses, see Suttys, et al.,Eur. J. Biochem., 195, 465 (1991); and to induce the production of IL-6or other growth factors, see Van Hinsbergh, et al., Blood, 72, 1467(1988)). TNF-α has also been also reported to participate, eitherdirectly or indirectly, in various diseases such as infectious diseasescarried by trypanosoma strains of the genus Plasmodium, see Cerami, A.,et al., Immunol. Today, 9, 28 (1988)); autoimmune diseases such assystemic lupus erythematosus (SLE) and arthritis, see Fiers, W., FEBS,285, 199 (1991); Acquired Immune Deficiency Syndrome (AIDS), see Mintz,M., et al., Am. J. Dis. Child., 143, 771 (1989); septicemia, see Tracey,K. J., et al., Curr. Opin. Immunol., 1, 454 (1989); and certain types ofinfections, see Balkwill, F. R., Cytokines in Cancer Therapy, OxfordUniversity Press (1989).

TNF-α and Inflammatory Response

Infection and tissue injury induce a cascade of biochemical changes thattrigger the onset of perplexing reactions of the immune system,collectively referred to as inflammatory response. The evolution of thisresponse is based, at least in part, on local vasodilation or enhancingvascular permeability and activation of the vascular endothelium, whichallows white blood cells to efficiently circulate and migrate to thedamaged site, thereby increasing their chances to bind to and destroyany antigens. The vascular endothelium is thought to then be activatedor inflamed. Generally, inflammation is a welcomed immune response to avariety of unexpected stimuli, and as such it exhibits rapid onset andshort duration (acute inflammation). Its persistent or uncontrolledactivity (chronic inflammation) has, however, detrimental effects to thebody and results in the pathogenesis of several immune diseases, suchas: septic shock, rheumatoid arthritis, inflammatory bowel diseases andcongestive heart failure. See “Tumor Necrosis Factors. The molecules andtheir emerging role in medicine” B. Beutler, Ed., Raven Press, N.Y.1992, pages 1-590.

The unfolding of an effective immune response typically requires therecruitment of a variety of cells and the orchestration of a series ofbiological events. This complex intercellular coordination andinteraction is mediated by a group of locally secreted low molecularweight proteins that are collectively called cytokines. These proteinsbind to specific receptors on the cell surface and triggersignal-transduction pathways that ultimately alter gene expression inthe target cells, thereby regulating an efficient inflammatory response.

Cytokines may exhibit properties of pleiotropism (a given protein exertsdifferent effects on different cells), redundancy (two or more cytokinesmediate similar functions), synergism (the combined effect of twocytokines is greater than the additive effect of each individualprotein) and antagonism (the effect of one cytokine inhibiting theeffect of another). To this end, some of the cytokines arepro-inflammatory (induce inflammation), while some others areanti-inflammatory (inhibit inflammation). The class of pro-inflammatorycytokines includes: interleukin-1 (IL-1), interleukin-6 (IL-6) and tumornecrosis Factor-alpha (TNF-α). See “Tumor Necrosis Factors. Themolecules and their emerging role in medicine” B. Beutler, Ed., RavenPress, N.Y. 1992, pages 1-590. These cytokines are secreted bymacrophages shortly after the initiation of the inflammatory responseand induce coagulation, increase the vascular permeability and activatethe expression of adhesion molecules on vascular endothelial cells (forexample, TNF-α stimulates expression of E-selection, that binds to andrecruits neutrophils to the site of damage). Subsequently, and during amore systemic immune response, these cytokines act on several organs ofthe body, including bone marrow and liver to ensure the increasedproduction of white blood cells and the synthesis of appropriatehormones and acute-phase proteins. In addition, they act on thehypothalamus and induce fever, which helps to inhibit the growth ofpathogens and enhances the overall immune reaction.

TNF-α and the Pathogenesis of Various Diseases and Conditions

As with any other cytokine, TNF-□ is neither completely beneficial norcompletely destructive to the host. Rather, balance of its productionand regulation is maintained to ensure that the host can effectivelyreact to invading microorganisms without compromising host well-being inthe process. Being a mediator of inflammation, TNF-∇ helps the body inits fight against bacterial infections and tissue injuries by boostingan appropriate immune response. However, its overproduction leads tochronic inflammation, has detrimental effects to the body and plays amajor role in the pathogenesis of several diseases, some of which aresummarized below.

Bacterial septic shock. This disease typically develops followinginfection by certain gram-negative bacteria, such as E. coli,Enterobacter aerogenes and Neisseria meningitidis. These bacteria bearon their cell walls certain lipopolysaccharides (endotoxins) thatstimulate macrophages to overproduce IL-1 and TNF-α, which in turn causethe septic shock. The symptoms of this condition, which are often fatal,include a drop in blood pressure, fever, diarrhea and widespread bloodclotting. In the United States alone, this condition afflicts about500,000 persons per year and causes more than 70,000 deaths. The annualcost for treating this disease is an estimated $ 5-10 billion.

Rheumatoid Arthritis. This is the most common human autoimmune disease,affecting about 1% of the Western population and is a major source ofdisability, which in its severe form leads to death. See Szekanecz, Z.;Kosh, A. E.; Kunkel, S. L.; Strieter, R. M. “Cytokines in rheumatoidarthritis. Potential targets for pharmacological applications”. ClinicalPharmacol. 1998, 12, 377-390. Camussi, G.; Lupin, E. “The future role ofanti-tumor necrosis factor products in the treatment of rheumatoidarthritis”. Drugs 1998, 55, 613-620. This condition is characterized byinflammation and cellular proliferation of the synovium, which resultsin the invasion of the adjacent cartilage matrix, its subsequent erosionand ultimately bone destruction. Although the origins of thisinflammatory response are poorly understood, an increased expression ofTNF-α and IL-1 have been found around the area of cartilage erosion.More recently, the pathogenic role of TNF-α in this disorder has beenextensively studied and experimentally verified. Furthermore, clinicaldata suggest that neutralization of TNF-α may be a therapeutic approachto reduce the erosive process. To date, however, current therapy, whileproviding temporary relief does not alter the fundamental mechanisms ofprogress or process of the disease.

Inflammatory bowel diseases and related conditions. This class ofdiseases which include Crohn's disease and ulcerative colitis aredebilitating disorders, characterized by chronic inflammation ofintestinal mucosa and lamina propria. Although the events that triggertheir onset are unknown, they are associated with significant leukocyteinfiltrate and local production of soluble mediators. TNF-α is thereforeconsidered to be a key mediator in the pathogenesis of these conditions,either by a direct cytotoxic action or as an orchestrator of theinflammatory cascade. See, for example, Armstrong, A. M.; Gardiner, K.R.; Kirk, S. J.; Halliday, M. J.; Rowlands, B. J. “Tumour necrosisfactor and inflammatory bowel disease”. Brit. J. Surgery 1997, 84,1051-1058. Data based on accepted animal models also supports therationale for a therapeutic study in human IBD, aimed at reducing theeffect of TNF. See Van Deventer, S. J. H. “Tumour necrosis factor andCrohn's disease” Gut, 1997, 40, 443.

Congestive heart failure. Activation of cytokines, and especially TNF-α,occurs in patients with chronic heart failure and acute myocardialinfarction. See Ferrari, R. “Tumor necrosis factor in CHF: a doublefacet cytokine”. Cardiovascular Res. 1998, 37, 554-559. Moreover, TNF-αhas been demonstrated to trigger the apoptotic process in cardiacmyocytes both directly (by binding to and genetically reprogrammingthese cells) and indirectly (through local NO production, which alsoleads to cell death).

HIV replication. Replication of HIV is activated by the inducibletranscription factor NF-κB, which in turn is induced by TNF-α. HIVexpression can be induced by TNF in macrophage lines and T-cell cloneschronically infected with the virus. Infusion of recombinant TNF in asmall number of patients with AIDS-related Kaposi's sarcoma appeared tocause an increase in the HIV p24 antigen level, a marker of viralreplicative activity. See “Therapeutic modulation of cytokines” CRCPress, Inc., N.Y. 1996, pages 221-236. These results provide amechanistic basis for considering the use of a TNF blocker to reduceinfectious HIV burden.

Other TNF mediated pathologies. There is an ever-increasing list ofconditions in which there is some evidence that TNF is involved.“Therapeutic modulation of cytokines” CRC Press, Inc., N.Y. 1996, pages221-236. In some cases, such as transplantation, graft-vs-host disease,and ischemia/reperfusion injury the potential mechanism of pathogenesisimplicates the pro-inflammatory activity of TNF-α to a variety of tissuecells. Others, such as the suppression of insulin responsiveness innon-insulin-dependent diabetes, relate to more selective actions ofTNF-α that appear to fall outside the standard pro-inflammatory model.TNF-α has been detected locally in patients afflicted with otitis media(inner ear infection, with or without effusion), see for example,Willett, D. N., Rezaee, R. P., Billy, J. M., Tighe, M. A., and DeMaria,T. F., Ann. Rhinol Laryngol, 107 (1998); Maxwell, K., Leonard, G., andKreutzer, D. L., Arch Otolarygol Head Neck Surg, vol. 123, p. 984(September 1997), and with sinusitis, see for example Nonoyana, T.,Harada, T., Shinogi, J., Yoshimura, E., Sakakura, Y., Auris NasusLarynx, 27(1), 51-58 (January 2000); Buehring I., Friedrich B., Schaff,J., Schmidt H., Ahrens P., Zielen S., CLin Exp Immul, 109(3), 468-472,Sep. 1997).

TNF-α and IL-1 Modulation as Therapeutic Approaches

Prior to the isolation of TNF-α, the employed therapeutic approaches tothe above diseases were targeting the reduction of chronic inflammationand were based on steroidal and non-steroidal anti-inflammatorytreatment. However, our recent understanding of TNF-α has led to thedevelopment of alternative strategies based on its selective inhibition.These general strategies are summarized below.

Steroidal treatment. This treatment, which includes the use ofcorticosteroids, causes the reduction in the number and the activity ofthe immune-system cells. The mechanism of action of the corticosteroidsinvolves crossing of the plasma membrane and binding on receptors in thecytosol. The resulting complexes are then transported to the cellnucleus where they bind to specific regulatory DNA sequences, therebydown-regulating the cytokine production. Although currently employed,this strategy has several disadvantages since it is not specific forTNF-α but also downregulates several other cytokines that may playimportant roles in an effective immune reaction. Moreover, use ofsteroids is also implicated with the development of cancer (for exampleprostate cancer).

Non-steroidal anti-inflammatory treatment. This strategy includes use ofcompounds such as aspirin that indirectly reduce inflammation. This isusually accomplished by inhibiting the cyclooxygenase pathway by whichprostaglandins and thromboxanes are produced. This action reduces thevascular permeability and provides temporary relief. To this end, thisstrategy does not regulate the production of cytokines and has little orno effect in diseases associated with chronic inflammation.

Engineered monoclonal anti-TNF antibodies. This strategy involves aselection of monoclonal antibodies that are capable of binding to andneutralizing TNF-α. Although the preliminary clinical studies have shownsome positive results, this approach is still in its infancy and notgenerally accepted. One of the problems to be addressed is that themonoclonal antibodies are of murine origin and in humans they elicitanti-immunoglobulin immune responses which limit their clinical use.Recombinant engineering techniques are being pursued to create“humanized” versions of the rodent antibodies that will maintainactivity against TNF-α and will be accepted more easily by the humanimmune system.

Use of soluble TNF-α receptors. The use of soluble receptors againstTNF-α is a new therapeutic approach. Although these receptors arecreated to bind and neutralize TNF-α, they also enhance its activity byprolonging its lifespan in blood circulation. Furthermore, the long termimmunological response to this type of treatment is beng evaluated.

Gene therapy. The goal of this approach is to decrease inflammation notby decreasing the expression of TNF-α but by increasing the localproduction of anti-inflammatory cytokines. The treatment consists ofdirect injection of cDNA expression vectors encoding foranti-inflammatory cytokines to the inflammed area, which couldantagonize the effect of TNF. The efficacy of this method is currentlyunder investigation in preclinical studies and its long term effects onthe immune response remain unknown.

Other disease stated and conditions. Additionally, TNF-α and/or IL-1have been more recently identified as participating in modulatingangiogenic vascular endothelial growth factor (VEGF), see E. M. Paleologet al., Arthritis & Rheumatism, 41, 1258 (1998), and may participate intuberculous pleurisy, rheumatoid pleurisy, and other immune disorders,see T. Söderblom, Eur. Respir. J., 9, 1652 (1996). TNF-α has also beenreported to effect expression of certain cancer cell genes for multidrugresistance-associated protein (MRP) and lung resistance protein (LRP),see V. Stein, J. Nat. Canc. Inst., 89, 807 (1997), and to participate inchronic and congestive heart failure, and related cardiovasculardisease, see for example R. Ferrari, Cardiovascular Res., 37, 554(1998); C. Ceconi et al., Prog. Cardiovascular Dis., 41, 25 (1998), andto either directly or indirectly mediate viral infection, see D. K.Biswas, et al., J. Acquired Immune Defic Syndr. Hum Retrovirol., 18,426-34 (1998) (HIV-1 replication); R. LeNauor, et al., Res. Virol., 145,199-207 (1994) (same); T. Harrer, et al., J. Acquir. Immune Defic.Syndr., 6, 865-71 (1993) (same); E. Fietz, et al., Transplantation, 58(6), 675-80 (1994) (human cytomegalovirus (CMV) regulation); D. F.Zhang, et al., Chin. Med. J., 106, 335-38 (1993) (HCV and HBVinfection). Furthermore, antagonists of TNF-α have also been shownuseful in the treatment of skin redness of a neurogenic origin. SeeEuropean Patent EPO-774250-B1 (to De Lacharriere et al.).

TNF-α has also been identified as expressed at heightened levels inhumans diagnosed as obese or exhibiting insulin resistance, and is thus,a modulator of diabetes. See Hotamisligil, G., Arner, P., Atkuinson, R.,Speigelman, B. (1995), “Increased adipose tissue expression of tumornecrosis Factor-α (TNF-α) in human obesity and insulin resistance. J.Clin. Invest. 95: 2409-2415. TNF-α has also been identified as animportant modulator of transplant rejection. See Imagawa, D., Millis,J., Olthoff, K., Derus, L., Chia, D., Sugich, L., Ozawa, M., Dempsey,R., Iwaki, Y., Levy, P., Terasaki, P., Busuttil, R. (1990) “The role oftumor necrosis factor in allograft rejection” Transplantation, vol. 50,No. 2, 219-225.

These observations highlight the importance and desirability ofidentifying novel strategies and/or novel compounds and classes ofcompounds that selectively influence the production of TNF-α and/orIL-1. Small molecules that selectively inhibit these cytokines aretherefore particularly medicinally and biologically important in, forexample, maintaining an active immune system and in treatinginflammation based diseases.

Preferred Methods of Synthesis of the Present Invention

Certain embodiments of the invention include novel methods of making thecompounds having the chemical structure of Formulae (II), (IIA), or(IIB), as well as novel methods of making known analogs of the known thecompounds having the chemical structure of Formulae (II), (IIA), or(IIB), for example, the compounds of Formulae (I) and (IA).

The compounds of the present invention, and specifically, the compoundshaving the chemical structure of Formula (II), (IIA), or (IIB), may beprepared either synthetically or semi-synthetically. If preparedsynthetically, commonly available starting materials may be used,including, but not limited to bicyclic compounds having reactive halidemoieties. The at-least-three-ringed compounds of the present inventionmay be synthesized according to various ring closure reactions. Suchreactions include, but are not limited to the Diels-Alder reaction, andthe Dieckmann Condensation reaction. The Diels-Alder reaction preferablyinvolves the reaction of a diene and an a substituted alkenyl moiety,such that the third ring of the desired compound is formed. TheDieckmann Condensation reaction may be preferably followed by thereduction of the resulting cycloketone moiety. Compounds of the presentinvention may be purified and isolated, following such synthetic methodsand other well-known synthetic methods, by use of procedures, such achromatography or HPLC, as well known to those skilled in the art.

Alternatively, according to the present invention, the compounds havingthe chemical structure of Formulae (I) and (IA), and certain specificanalogs and derivatives thereof, may be extracted and isolated, at leastin the form of a crude extract comprising acanthoic acid, from the rootbark of Acanthopanax koreanum Nakai. Such an extract may preferably beproduced according to the following method:

Approximately one kilogram of dried root bark of A. koreanum Nakai isobtained, chipped, and covered with between 1 L to 3 L, and preferably 2L, of a suitable solvent, most preferably methanol. This mixture ismaintained at a temperature ranging from 20° to 60°, and may bemaintained at room temperature, for at least 10 hours, and preferablyfor 12 hours. The mixture is then filtered to remove and retain thefiltrate. This procedure is repeated, preferably at least two additionaltimes, and the combined filtrates are concentrated under a reducedpressure to obtain an extract.

Approximately 100 grams of the extract is partitioned with 200 mL to 400mL, preferably 300 mL, of an aqueous solution, preferably water and 200mL to 400 mL, preferably 300 mL, of an organic solution, preferablydiethyl ether. The organic fraction is separated therefrom and thenconcentrated under a reduced pressure to obtain a further extract. Saidfurther extract is purified, preferably by column chromatography andeven more preferably by use of a silica gel column, using a mixture ofsuitable organic solvents, preferably hexane and ethyl acetate as aneluent to obtain isolated acanthoic acid.

This isolated compound of Formulae (I) and (IA) may then bysynthetically modified to yield certain compounds of the presentinvention, specifically the compounds having the chemical structure ofFormula (II) or (IIA). For example, ester R₁ analogs of acanthoic acidmay be formed according to acid-catalyzed nucleophilic addition of analkyl alcohol to the carboxylic acid moiety of acanthoic acid. Ether R₁analogs of acanthoic acid may be formed from either primary alkylhalides or alcohols according to the Williamson Ether synthesis, or viathe reduction of a primary alcohol moiety. Alkyl, alkenyl, and alcoholicR₁₀ analogs of acanthoic acid may be formed via catalytic hydrogenationof the alkenyl group, or via electrophilic addition of, preferably, HClor HBr or other suitable alkyl halides. Substitution analogs at theother R positions of acanthoic acid may be formed by displacementreactions involving alkyl halides, provided suitable reactive groups andrelated protecting groups are employed to encourage the desiredreaction. According to these reaction and other well-known syntheticreactions, the production of the full range of the compounds of thepresent invention, given the description of those compounds providedherein, is within the skill of those of the art.

Fully-synthetic approaches for the preparation of the compounds of theinvention, including compounds of general Formulae (I), (IA), (II),(IIA), and (IIB) are described herein. This synthesis includes one ormore retrosynthetic analyses of acanthoic acid and its analogs,syntheses of radioactively labeled acanthoic acid and its analogs,syntheses of dimers and conjugates of the compounds of general Formulae(I), (IA), (II), (IIA), and (IIB). Those of skill in the art will alsoappreciate that these approaches are also fully applicable to thepreparation of kauranoic acid and its analogs.

The Compound of Formula (I) and its Naturally-Occuring Analogs

The root bark of Acanthopanax koreanum Nakai (Araliaceae), which isfound indigenously in Cheju Island, The Republic of Korea, has been usedtraditionally as a tonic and sedative, as well as a remedy for thetreatment of rheumatism and diabetes. During their investigation of thisfolk medicine, Chung and coworkers identified from its pharmacologicallyactive extracts two novel tricyclic diterpenes: acanthoic acid(Compound 1) and its methyl ester (Compound 2), as depicted in FIG. 1.See Kim, Y. H.; Chung, B. S.; Sankawa, U. “Pimaradiene diterpenes fromAcanthopax Koreanum”. J. Nat. Prod. 1988, 51, 1080-1083. Acanthoic acidis a pimarane (3). However, in sharp contrast to the other members ofthe pimaranes family, 1 is distinguished by an unusual stereochemicalrelationship between the C8 and C10 centers that provides a unique modeof connectivity of the BC ring system.

Prior to this invention, no complete chemical synthesis existed forproduction of the chemical having the structure of Formula (I) or itsanalogs. Importantly, the chemical structure of Formula (I), 1, (FIG. 1)possesses a biological profile as an anti-inflammatory agent. Morespecifically, in vitro studies with activated (inflammed)monocytes/macrophages revealed that treatment with 1 (approximately 0.1to approximately 1.0 microgram/ml for 48 hours) leads to anapproximately 90% inhibition of the TNF-α and IL-1 production. Thisinhibition was concentration dependent and cytokine-specific, sinceunder the same conditions the production of IL-6 or IFN-γ(interferon-gamma) were not affected. The in vivo effects of acanthoicacid were evaluated in mice suffering from silicosis (chronic lunginflammation) and cirrhosis (liver inflammation and hepatic fibrosis).Histologic analysis revealed that treatment with compound 1 led to asubstantial reduction of fibrotic granulomas and a remarkable recoveryof the cirrotic liver cells. These dramatic results can be attributed,at least partially, to inhibition of pro-inflammatory cytokines, such asTNF-α and IL-1, mediated by 1. Compound 1 also shows very littletoxicity in mice and only upon orally administering a high concentration(LD>300 mg/100 g of body weight). See Kang, H.-S.; Kim, Y.-H.; Lee,C.-S.; Lee, J.-J.; Choi, I.; Pyun, K.-H., Cellular Immunol. 1996, 170,212-221. Kang, H.-S.; Song, H. K.; Lee, J.-J.; Pyun, K.-H.; Choi, I.,Mediators Inflamm. 1998, 7, 257-259.

The chemical structure of Formula (I) thus has potent anti-inflammatoryand anti-fibrotic effects and reduces the expression of TNF-α and IL-1.Acanthoic acid is thus used as a chemical prototype for the developmentof the novel compounds of the invention.

Retrosynthetic Analyses of the Compounds of Formulae (I), (II) and (IIB)

The compounds of Formulae (I), (II) and (IIB), and preferably thecompound of Formula (I) and compounds of Formulae (IIB) designated TTL1,TTL2, TTL3, and TTL4 herein, the may be synthesized according to anaspect of the invention. The bond disconnections of the compounds ofFormulae (I) are shown in FIG. 2. The novel structural arrangement ofthe BC rings and the presence of the quaternary C13 center constitute anunusual motif and lead to a novel strategy that is oneaspect of theinvention. This motif is fixed, in one step, into the desiredstereochemistry by employing a Diels-Alder methodology. A diene, forexample, 14, and a dienophile, such as 15 (Y: oxazolidinone-basedauxiliary), were identified as the appropriate starting materials for anendo selective Diels-Alder reaction. To further ensure the desiredregiochemical outcome of this cycloaddition, diene 14 was functionalizedtransiently with a heteroatom (for example, X═OTBS or SPh), which willbe subsequently removed from the product 13. The generally observed endopreference of this reaction was used to predict the stereochemicalrelationship between the C12 and C13 centers as shown in product 13,while the diastereofacial seletivity of the process will be controlledeither by a chiral auxiliary at the carbonyl center of the dienophile orby using a chiral catalyst. See Xiang, A. X.; Watson, D. A.; Ling. T.;Theodorakis, E. A. “Total Synthesis of Clerocidin via a Novel,Enantioselective Homoallenylboration Methodology”. J. Org. Chem. 1998,63, 6774-6775.

Diene 14 may be formed by a palladium (0) catalyzed construction of theC8-C11 bond, revealing ketone 16 as its synthetic progenitor. Thisketone was formed from the known Wieland-Miescher ketone (17), which inturn was readily available by condensation of methyl vinyl ketone (19)with 2-methyl 1,3-cyclohexane dione (18) (FIG. 2).

In one aspect of the invention, it is recognized that thefunctionalities and relative stereochemistry of the AB ring system ofacanthoic acid (1) are akin to those in the structure of podocapric acid(20). See “The total synthesis of natural products.” ApSimon, Ed.; JohnWiley & Sons, Inc., 1973, Volume 8, pages 1-243. Among the severalsynthetic strategies toward 20, highlights of the ones may be arerelevant to our proposed synthesis of 1 are shown in FIG. 5. Accordingto the invention, these approaches allowed the prediction of thestereochemical outcome of the synthesis of The compounds of Formulae(I), (II) and the contrary stereochemical of the compounds of Formulae(IIB), and the compounds of Formulae (IIB) that are designated TTL1,TTL2, TTL3, and TTL4 herein.

Complete syntheses of the Compounds of Formulae (I), (II) and (IIB)

The initial step of the synthesis of acanthoic acid (1), and of allcompounds of Formulae (I), (II) and (IIB), involves the reaction of aWieland-Miesher ketone (17). This compound was readily available fromcompounds 18 and 19 as a single enantiomer by a Michaeladdition/Robinson annulation sequence using catalytic amounts of(R)-proline. Selective protection of the more basic C9 carbonyl group of17, followed by a reductive alkylation of enone 34 with methylcyanoformate gave rise to ketoseter 36. Transformation of 36 to 39 wasbased on previous studies, see Welch, S. C.; Hagan, C. P. “A newstereoselective method for developing ring A of podocapric acidcompounds” Synthetic Commun. 1972, 2, 221-225, as depicted in FIG. 3.Reduction of the ester functionality of 39, followed by silylation ofthe resulting alcohol and acid-catalyzed deprotection of the ketal unitthen afforded ketone 40. Conversion of 40 to the desired diene 42 wasaccomplished by a two step sequence involving transformation of 40 toits corresponding enol triflate derivative, followed by palladiumcatalyzed coupling with vinyl stannane 41. See Farine, V.; Hauck, S. I.;Firestone, R. A. “Synthesis of cephems bearing olefinic sulfoxide sidechains as potential b-lactamase inhibitors” Bioorg. & Medicinal Chem.Lett. 1996, 6, 1613-1618.

The steps that were used in the completion of the synthesis of acanthoicacid (1), and are used in the completion of the syntheses of compoundsof Formulae (I), (II) and (IIB) are depicted in FIG. 5, as Scheme 2. ADiels-Alder cycloaddition between diene 42 and dienophile 43, followedby reductive desulfurization with Raney Ni produces the tricyclic system44 with the desired stereochemistry. Transformation of 44 to the Weinrebamide, followed by reduction with DIBALH generated aldehyde 45, whichupon Wittig reaction gave rise to olefin 46. Fluoride-induceddesilylation of 46, followed by a two steps oxidation of the resultingalcohol to the carboxylic acid produced acanthoic acid (1), and may beused to produce the compounds of Formulae (I), (II) and (IIB) byappropriate substitution of the intermediates.

One important step to the synthesis of compounds of Formulae (I) and(IA), and the compounds of Formulae (II), (IIA) and (IIB), is theDiels-Alder reaction. This reaction, and the use and selection of one ormore appropriately substituted dienes and/or dienophiles permits theselective synthesis of compounds of Formula (II) or the selectivesynthesis of compounds of Formula (IIB). For example, the followingpreferred dienophiles may be used in place of the dienophiles, forexample, compound 43 and pimarane (103), as depicted herein in, forexample, FIGS. 5, 7, 8, 21, and 23, as Reaction Schemes 2, 3, 4, 5, and6, to selectively yield compounds of Formulae (II) and (IIB). Exemplarydienophiles include those of Formulae (III):

-   -   wherein the numbered R-groups (R₉, R₁₄, and R₁₅) are as        designated above for the compounds of Formula (IIB), and the        unnumbered R groups may be any of R₁ through R₁₅ as designated        above for the compounds of Formula (IIB).

Furthermore, the electronic conformation of the diene, for examplecompound (42) and compound (112), as depicted herein in, for example,FIGS. 5, 7, 8, 21, and 23, as Reaction Schemes 2, 3, 4, 5, and 6,respectively, may be altered by the covalent linkage ofelectron-donating or electron-withdrawing group, for example, pHS, tothe diene. As exemplified herein, such a covalently linkedelectron-donating or electron-withdrawing group effects the orientationof the incoming dieneophile.

Thus, according to one aspect of the invention, the chiral nature ofdiene 42 allows it to be used to induce asymmetry during thecycloaddition. Examination of a minimized model of 42 indicates that theangular methyl at C10 influences the facial selectivity of the reactionand allow more efficient approach of the dienophile from the top face ofthe diene. This approach produced the adduct that leads to compounds ofFormulae (IIB). This approach also allowed for the development of acatalytic asymmetric variant of the Diels Alder reaction. The benefitsof using chiral catalysts, as opposed to chiral auxiliaries, are obviousand well documented in the recent literature.

One preferred embodiment of the invention is the use of catalyst 49,that was developed and applied by Corey toward an improved asymmetricsynthesis of cassiol (Scheme 3) See Corey, E. J.; Imai, N.; Zhang, H.-Y.J. Am. Chem. Soc. 1994, 116, 3611. Compound 49 was shown to allowDiels-Alder cycloaddition of an electronically rich diene 47 withmethacrolein (48) and produce exclusively the endo adduct in excellentyield and enantiomeric excess (83% yield, 97% ee).

Application of the above methodology to our synthesis is depicted inFIG. 8, as Scheme 4, Use of catalyst 49 provided additional versatilityand significantly shorten the total amount of steps required forcompletion of the total synthesis of 1.

Synthesis of Radiolabeled Compounds of Formula (I)

A radiolabeled sample of a compound of Formulae (I), (II), (IIA) or(IIB) may be synthesized and is useful in pharmacological andpharmacokinetic studies, For example, a C14-labeled methylene carbon isincorporated on the compound of Formulae (I) using aldehyde 52 as astarting material (as depicted in FIG. 4, Scheme 4). The C14-labeledyield, required for the Wittig chemistry, is prepared in two steps fromC14-labeled iodomethane and triphenyl-phosphine, followed by treatmentwith a base, such as NaHMDS. Base-induced deprotection of themethylester produces radiolabeled a compound of Formulae (I), (II),(IIA) or (IIB).

Objectives of the Syntheses of the Compounds of Formula (II), (IIA) and(IIB)

One aspect of the invention is the identification of novelanti-inflammatory drugs having the structure of the compounds of Formula(II), (IIA) and (IIB). Biological screening of synthetic intermediatesand rationally designed compounds of Formula (II) provide informationand guide the design requirements.

The design and synthesis of analogs of the compounds of Formula (II) isbased on the following objectives: (a) defining the minimum structuraland functional requirements the compounds of Formula (II) that areresponsible for the TNF-α and IL-1 modulating activity (minimumpharmacophore); (b) improving the TNF-α and IL-1 modulating activity ofthe compounds of Formula (II) by altering the structure, particularlythe R-groups of the minimum pharmacophore (for example, SAR studies andmolecular recognition experiments); (c) examining the mode of action ofthe compounds of Formula (II) by photoaffinity labeling studies; (d)modifying and improving the solubility and membrane permeability of thecompounds of Formula (II); (e) synthesizing and study dimers orconjugates of the compounds of Formula (II); selective delivery unitsand (f) redesigning and refining the target structure by evaluating theobtained biological data.

Of particular significance to the rational design of novel the compoundsof Formulae (II), (IIA) and (IIB) are the recent reports thatmodification of the A and C rings of oleanolic acid (53), as depicted inFIG. 9, lead to enhanced antiproliferative and antiinflammatoryactivity. See Honda, T.; Rounds, B. V.; Gribble, G. W.; Suh, N.; Wang,Y.; Sporn, M. B. “Design and synthesis of2-cyano-3,12-dioxolean-1,9-dien-28-oic acid, a novel and highly activeinhibitor of nitric oxide production in mouse macrophages” Biorg. &Medic. Chem. Lett. 1998, 8, 2711-2714. Suh, N. et al “A novel syntheticoleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid,with potent differentiating, antiproliferative and anti-inflammatoryactivity” Cancer Res. 1999, 59, 336-341. More specifically, SAR studieswith commercially available 53 and semisynthetic derivatives thereofhave led to the recognition that that: (a) attachment ofelectron-withdrawing groups, such as nitrile, at the C2 positionincreases the biological potency of 53 (FIG. 9); (b) an α,β unsaturatedketone functionality at the C ring is a strong enhancer of potency. Thecombination of these observations lead to the semisynthesis of adesigned triterpenoid 54 (FIG. 9), shown to be 500-fold more active thanany other known triterpenoid in suppressing the inflammatory enzymesiNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2)(FIG. 9).

Synthesis of the Compounds of Formulae (II), (IIA) and (IIB)

The thirteen-step synthesis of the compounds of Formulae (II), (IIA) and(IIB) (as shown in FIGS. 4 and 8, Schemes 1 and 4, respectively) isefficient and as such, it allows the preparation of a variety of analogsuseful in SAR studies. The biological significance of the unusualtricyclic scaffold of the compounds of Formulae (II), (IIA) and (IIB)(the C8 epimer is constructed using the appropriate Diels-Aldercatalyst). The sites that are easily altered via the synthetic approachof the invention, or by standard modifications of our syntheticintermediates are shown in the FIG. 10, and representative examples ofthe compounds of Formula (II) are shown in FIG. 11.

The desired chemical scaffold of the compounds of Formula (II), (IIA)and (IIB) may also be incorporated into solid support such as, forexample, a Wang resin. This permits the facile construction ofcombinatorial libraries of the compounds of Formula (II), (IIA) and(IIB). Furthermore, according to the invention, preferred TNF-α and IL-1modulators may be more rapidly identified and screen that currentlypossible.

Photoaffinity Labeling Studies.

The backbone of the compounds of Formula (II), (IIA) and (IIB) is alsopreferably labeled with a reactive cross-linker, that is useful inphotoaffinity labeling studies. These studies assist in theidentification of the in vivo target of the compounds of Formula (II),(IIA) and (IIB) and provide fundamental insights into the mode of actionof acanthoic acid and on the activation of TNF-α. The C19 carboxylicacid or the C15 aldehyde (precursor of 1) are useful in cross-linkingexperiments with the appropriate photosensitive reagents (see 60 and 61,FIG. 12).

Synthesis of Dimers and Conjugates of the Compounds of Formula (II),(IIA) and (IIB)

Dimeric forms the compounds of Formula (II), (IIA) and (IIB), such asfor example 62 (n=1), have been isolated from natural sources and,furthermore, the dexamethasone-acanthoic acid conjugate 63 providesbiologically interesting results toward a drug targeting a steroidreceptor with potential implications in cancer research. See Chamy, M.C.; Piovano, M.; Garbarino, J. A.; Miranda, C.; Vicente, G.Phytochemistry 1990, 9, 2943-2946. While no biological studies of thisclass of compounds has been performed, according to the invention,dimeric analogs of Formula (II), (IIA) and (IIB) are evaluated.Synthetic acanthoic acid or bioactive analogs of 1 are used as monomericpartners and their coupling is performed using standard techniques,included those described herein.

Experimental Techniques

All reactions were carried out under an argon atmosphere in dry, freshlydistilled solvents under anhydrous conditions, unless otherwise noted.Tetrahydrofuran (THF) and diethyl ether (Et₂O) were distilled fromsodium/benzophenone; dichloromethane (CH₂Cl₂), hexamethyl phosphoramide(HMPA), and toluene from calcium hydride; and dimethyl formamide (DMF)from calcium chloride. Yields refer to chromatographically andspectroscopically (¹H NMR) homogeneous materials, unless otherwisestated. Reagents were purchased at highest commercial quality and usedwithout further purification, unless otherwise stated. Reactions weremonitored by thin-layer chromatography carried out on 0.25 mm E. Mercksilica gel plates (60F-254) using UV light as visualizing agent and 7%ethanolic phosphomolybdic acid, or p-anisaldehyde solution and heat asdeveloping agents. E. Merck silica gel (60, particle size 0.040-0.063mm) was used for flash chromatography. Preparative thin-layerchromatography separations were carried out on 0.25 or 0.50 mm E. Mercksilica plates (60F-254). NMR spectra were recorded on a Varian 400and/or 500 Mhz instruments and calibrated using a residual undeuteratedsolvent as an internal reference. The following abbreviations were usedto explain the multiplicities: s=singlet; d=doublet, t=triplet;q=quartet, m=multiplet, b=broad. IR spectra were recorded on a NicoletAvatar 320 FT-IR spectrometer. Optical rotations were recorded on aPerkin Elmer 241 polarimeter. High resolution mass spectra (HRMS) wererecorded on a VG 7070 HS mass spectrometer under chemical ionization(CI) conditions or on a VG ZAB-ZSE mass spectrometer under fast atombombardment (FAB) conditions.

Triketone 2. A solution of diketone 1 (50 g, 0.40 mol) in ethyl acetate(500 ml) was treated with triethylamine (72 ml, 0.52 mol) and methylvinyl ketone (36 ml, 0.44 mol). The reaction mixture was refluxed at 70°C. for 10 h and then cooled to 25° C. The solvent was removed underpressure and the resulting crude material was chromatographed directly(10-40% ether in hexanes) to yield triketone 2 (61 g, 0.31 mol, 78%). 2:colorless oil; R_(f)=0.25 (silica, 50% ether in hexanes); ¹H NMR (400MHz, CDCl₃) δ 2.75-2.59 (m, 4H), 2.34 (t, 2H, J=7.2 Hz), 2.10 (s, 3H),2.07-2.05 (m, 3H), 1.98-1.94 (m, 1H), 1.24 (s, 3H).

Wieland-Miescher ketone (3) A solution of triketone 2 (61 g, 0.31 mol)in dimethyl sulfoxide (400 ml) was treated with finely groundedD-proline (1.7 g, 0.01 mol). The solution was stirred at 25° C. for 4days and then stirred at 40° C. for 1 more day. The resulting purplecolored solution was cooled to 25 C, diluted with water (300 ml) andbrine (100 ml), and poured into a separatory funnel. The mixture wasextracted with ethyl ether (3×800 ml). The organic layers wereconcentrated (without drying) and subjected to chromatography (10-40%ether in hexanes) to give 59 g of a crude reddish-violet oil. Thematerial was again subjected to chromatography (10-40% ether in hexanes)and concentrated to yield 57 g of a yellow oil. The oil was dissolved inethyl ether (400 ml) and kept at 4° C. for 30 min, after which time alayer of hexanes (100 ml) was added on top of the ether. The two-layeredsolution was seeded with a few crystals and placed into a freezer (−28°C.) overnight. The resulting crystals were collected by filtration,rinsed with ice-cold hexanes (2×100 ml), and dried under pressure.Concentration of the mother liquor afforded another crop, and combiningthe crystals afforded the Wieland-Miescher ketone (3) (43 g, 0.24 mol,78%). 3: tan crystals; R_(f)=0.25 (silica, 50% ether in hexanes);[α]²⁵D: −80.0 (c=1, C₆H₆); ¹H NMR (400 MHz, CDCl₃) δ 5.85 (s, 1H),2.72-2.66 (m, 2H), 2.51-2.42 (m, 4H), 2.14-2.10 (m, 3H), 1.71-1.68 (m,1H), 1.44 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 210.7, 198.0, 165.6,125.7, 50.6, 37.7, 33.7, 31.8, 29.7, 23.4, 23.0.

Acetal 4. A solution of ketone 3 (43 g, 0.24 mol) in benzene (700 ml)was treated with p-toluenesulfonic acid (4.6 g, 0.024 mol) and ethyleneglycol (15 ml, 0.27 mol). The reaction was refluxed with a Dean-Starkapparatus and condenser at 120 C. Once water stopped collecting in theDean-Stark apparatus, the reaction was complete (approx. 4 h). Leavingthe reaction for longer periods of time tended to darken the reactionmixture and lower the overall yield. The reaction was cooled to 25° C.,quenched with triethylamine (5 ml, 0.036 mol), and poured into aseparatory funnel containing water (300 ml) and saturated sodiumbicarbonate (200 ml). The resulting mixture was then extracted withether (3×800 ml). The organic layers were combined, dried over MgSO₄,concentrated, and subjected to chromatography (10-40% ether in hexanes)to afford acetal 4 (48 g, 0.22 mol, 90%). 4: yellow oil; R_(f)=0.30(silica, 50% ether in hexanes); [α]²⁵D: −77 (c=1, C₆H₆); IR (film)υ_(max) 2943, 2790, 1667, 1450, 1325, 1250; ¹H NMR (400 MHz, CDCl₃) d5.80 (s, 1H), 3.98-3.93 (m, 4H), 2.43-2.35 (m, 3H), 2.34-2.20 (m, 3H),1.94-1.82 (m, 1H), 1.78-1.60 (m, 3H), 1.34 (s, 3H); ¹³C NMR (100 MHz,CDCl₃) δ 198.9, 167.5, 125.5, 112.2, 65.4, 65.1, 45.1, 34.0, 31.5, 30.1,26.9, 21.8, 20.6.

Ketoester 5. A solution of lithium (0.72 g, 0.10 mol) in liquid ammonia(400 ml) at −78 C was treated dropwise with a solution of acetal 4 (10g, 0.045 mol) and tert-butyl alcohol (3.7 ml, 0.045 mol) in ether (40ml). The resulting blue mixture was allowed to warm and stir at reflux(−33° C.) for 15 min and then cooled to −78° C. again. Sufficientisoprene (approx. 8 ml) was added dropwise to discharge the residualblue color of the reaction mixture. The reaction was then warmed in awater bath (50° C.) and the ammonia quickly evaporated under a stream ofdry nitrogen. The remaining ether was removed under pressure to leave awhite foam. After a further 5 min under high vacuum, the nitrogenatmosphere was restored, and the lithium enolate was suspended in dryether (150 ml) and cooled to −78° C. Methyl cyanoformate (4.0 ml, 0.050mol) was then added and the reaction stirred for 40 min at −78 C. Thereaction was warmed to 0° C. and stirred for 1 h more. Water (300 ml)and ether (200 ml) were added and the mixture poured into a separatoryfunnel containing saturated sodium chloride (100 ml). After separatingthe organic layer, the aqueous phase was extracted with ether (2×400ml). The combined organic layers were dried over MgSO₄, concentrated,and subjected to chromatography (10-40% ether in hexanes) to affordketoester 5 (7.0 g, 0.025 mol, 55%). 5: white powdery precipitate;R_(f)=0.40 (silica, 50% ether in hexanes; [α]²⁵D: −2.9 (c=1, C₆H₆); IR(film) ν_(max) 2943, 1746, 1700; ¹H NMR (400 MHz, CDCl₃) δ 4.00-3.96 (m,2H), 3.95-3.86 (m, 2H), 3.74 (s, 3H), 3.23 (d, 1H, J=13.2 Hz), 2.50-2.42(m, 3H), 2.05-1.92 (m, 1H), 1.79-1.50 (m, 5H), 1.32-1.28 (m, 2H), 1.21(s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 205.4, 170.0, 111.9, 65.2, 65.1,59.9, 52.0, 43.7, 41.6, 37.5, 30.3, 29.8, 26.2, 22.5, 14.0; HRMS, calcdfor C₁₅H₂₂O₅ (M+Na⁺) 305.1359, found 305.1354.

Ester 6. A solution of ketoester 5 (7.0 g, 0.025 mol) in HMPA (50 ml)was treated with sodium hydride (0.71 g, 0.030 mol). After stirring for3 h at 25° C., the resulting yellow-brown reaction mixture was quenchedwith chloromethyl methyl ether (2.3 ml, 0.030 mol) and the reactionallowed to stir an additional 2 h at 25° C. The resulting white-yellowmixture was then poured into a separatory funnel containing ice-water(100 ml), saturated sodium bicarbonate (50 ml), and ether (200 ml).After the layers were separated, the aqueous layer was extracted withether (3×200 ml). The combined ethereal extracts were dried over MgSO₄,concentrated, and subjected to chromatography (silica, 10-40% ether inhexanes) to yield ester 6 (7.7 g, 0.024 mol, 95%). 6: yellow oil;R_(f)=0.45 (silica, 50% ether in hexanes); [α]²⁵D: +26.3 (c=1, C6H6); IR(film) ν_(max) 2951, 1728, 1690, 1430, 1170; ¹H NMR (400 MHz, CDCl₃) δ4.89 (dd, 2H, J=22.8, 6.4 Hz), 3.93-3.91 (m, 2H), 3.90-3.84 (m, 2H),3.69 (s, 3H), 3.40 (s, 3H), 2.72-2.68 (m, 1H), 2.24 (bs, 2H), 1.80-1.42(m, 4H), 1.37-1.15 (m, 2H), 0.960 (s, 3H), 0.95-0.80 (m, 2H); ¹³C NMR(100 MHz, CDCl₃) δ 167.8, 150.5, 115.8, 112.1, 93.0, 65.2, 65.1, 56.3,51.3, 40.7, 40.3, 30.3, 26.4, 23.6, 22.9, 22.3, 13.9; HRMS, calcd forC₁₇H₂₆O₆ (M+Na⁺) 349.1622, found 349.1621.

Acetal 7. A solution of lithium (1.1 g, 0.17 mol) in liquid ammonia (400ml) at −78 C was treated dropwise with a solution of ester 6 (7.7 g,0.024 mol) in 1,2-DME (30 ml). The blue reaction mixture was allowed towarm and stir at reflux (−33° C.) for 20 min. The reaction mixture wasthen cooled to −78° C. again and rapidly quenched with excessiodomethane (15 ml, 0.24 mol). The resulting white slurry was allowed tostir at reflux (−33° C.) for 1 h, after which time the reaction waswarmed in a water bath (50° C.) with stirring for 1 h, allowing theammonia to evaporate. The reaction mixture was quenched with water (100ml), sodium bicarbonate (100 ml), and ether (200 ml) and poured into aseparatory funnel. After the layers were separated, the aqueous layerwas extracted with ether (3×200 ml). The combined ethereal extracts weredried over MgSO₄, concentrated, and subjected to chromatography (silica,10-30% ether in hexanes) to yield acetal 7 (4.1 g, 0.014 mol, 61%). 7:semi-crystalline yellow oil; R_(f)=0.80 (silica, 50% ether in hexanes);[α]²⁵D: +16.9 (c=10, C₆H₆); IR (film) ν_(max) 2934, 1728, 1466, 1379,1283, 1125, 942; ¹H NMR (400 MHz, CDCl₃) δ 3.95-3.80 (m, 4H), 3.64 (s,3H), 2.17-2.15 (m, 1H), 1.84-1.37 (m, 11H), 1.16 (s, 3H), 1.05-1.00 (m,1H), 0.87 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 177.7, 112.9, 65.2, 64.9,51.2, 44.0, 43.7, 38.1, 30.7, 30.3, 28.8, 23.4, 19.1, 14.7; HRMS, calcdfor C₁₆H₂₆O₄ (M+H⁺) 283.1904, found 283.1904.

Ketone 8. A solution of acetal 7 (4.1 g, 0.014 mol) in THF (50 ml) wastreated with 1M HCl dropwise (approx. 15 ml) at 25° C. with stirring.The reaction was monitored by thin layer chromatography and neutralizedwith sodium bicarbonate (30 ml) once the starting material disappeared.The resulting mixture was poured into a separatory funnel containingwater (100 ml) and ether (100 ml). After the layers were separated, theaqueous layer was extracted with ether (3×100 ml). The combined etherealextracts were dried over MgSO₄, concentrated, and subjected tochromatography (silica, 10-20% ether in hexanes) to yield ketone 8 (3.3g, 0.014 mol, 95%). 8: white crystals; R_(f)=0.70 (silica, 50% ether inhexanes); [α]²⁵D: +3.5 (c=1.0, C₆H₆); IR (film) ν_(max) 2943, 1728,1449, 1239, 1143, 1095, 985; ¹H NMR (400 MHz, CDCl₃) δ 3.62 (s, 3H),2.55-2.45 (m, 1H), 2.92-1.95 (m, 5H), 1.8-1.6 (m, 2H), 1.50-1.30 (m,4H), 1.14 (s, 3H), 0.98-0.96 (m, 1H), 0.90 (s, 3H); ¹³C NMR (100 MHz,CDCl₃) δ 214.8, 177.0, 54.4, 51.3, 49.3, 44.2, 37.9, 37.7, 33.1, 28.6,26.4, 22.8, 18.8, 17.0; HRMS, calcd for C₁₄H₂₂O₃ (M+Na⁺) 261.1461, found261.1482.

Alkyne 9. A solution of ketone 8 (2.0 g, 8.3 mmol) in ether (50 ml) wastreated with lithium acetylide (0.40 g, 13 mmol). The reaction wasstirred at 25 C for 1 h and then quenched with sodium bicarbonate (20ml) and water (30 ml). The mixture was poured into a separatory funneland the layers were separated. The aqueous layer was extracted withether (3×50 ml). The organic layers were combined, dried with MgSO₄,concentrated, and subjected to chromatography (silica, 10-30% ether inhexanes) to afford alkyne 9 (2.0 g, 7.6 mmol, 90%). 9: white solid;R_(f)=0.65 (silica, 50% ether in hexanes); ¹H NMR (400 MHz, CDCl₃) δ3.64 (s, 3H), 2.56 (s, 1H), 2.18-2.10 (m, 1H), 1.92-1.40 (m, 12H), 1.18(s, 3H), 1.17-1.01 (m, 1H), 0.81 (s, 3H); ¹³C NMR (100 MHz, CDCl₃)177.6, 86.8, 76.5, 75.0, 51.2, 50.5, 43.9, 52.5, 37.9, 35.3, 33.4, 28.8,23.5, 22.5, 19.1, 11.5; HRMS, calcd for C₁₆H₂₄O₃ (M+H⁺—H₂O) 247.1693,found 247.1697.

Alkene 10. A solution of alkyne 9 (0.50 g, 1.9 mmol) in 1,4 dioxane (20ml) and pyridine (2 ml) was treated with Lindlar's catalyst (100 mg).The mixture was hydrogenated under pressure (30 lbs/in²) for 7 min. Thereaction mixture was then diluted with ether (10 ml), filtered through apad of celite, and washed with ether (2×50 ml). The solvent wasevaporated under reduced pressure to afford alkene 10 (0.48 g, 1.8 mmol,95%). 10: colorless oil; ¹H NMR (400 MHz, CDCl₃) δ 6.58 (dd, 1H), 5.39(d, 1H), 5.14 (d, 1H), 3.64 (s, 3H), 2.20-2.11 (m, 2H), 1.93-1.65 (m,4H), 1.61 (s, 2H), 1.52-1.25 (m, 4H), 1.19 (s, 3H), 1.17-0.90 (m, 2H),0.89 (s, 3H).

Diene 11. A solution of alkene 10 (0.48 g, 1.8 mmol) in benzene (80 ml)and THF (20 ml) was treated with boron trifluoride etherate (1 ml, 7.9mmol), and the reaction mixture was refluxed at 100 C for 5 h. Aftercooling, the reaction was quenched with 1N NaOH (1 ml, 26 mmol) and themixture was poured into a separatory funnel containing water (100 ml)and ether (100 ml). After separating the layers, the aqueous layer wasextracted with ether (3×100 ml). The organic layers were combined, driedwith MgSO₄, concentrated, and subjected to chromatography (silica, 5%ether in hexanes) to afford diene 11 (0.42 g, 1.7 mmol, 95%). 11:colorless oil; R_(f)=0.95 (silica, 50% ether in hexanes); ¹H NMR (400MHz, CDCl₃) δ 6.26-6.23 (dd, 1H), 5.70 (s, 1H), 5.253 (d, 1H, J=19.2Hz), 4.91 (d, 1H, J=12.8 Hz), 3.64 (s, 3H), 2.22-2.12 (m, 2H), 2.10-1.94(m, 2H) 1.92-1.67 (m, 3H), 1.60-1.44 (m, 3H), 1.378 (d, 1H, J=13.6),1.21 (s, 1H), 1.19-1.00 (m, 2H), 0.86 (s, 3H); ¹³C NMR (100 MHz, CDCl₃)δ 177.7, 146.7, 136.1, 121.9, 113.3, 53.0, 51.2, 43.9, 38.0, 37.9, 37.4,28.5, 27.8, 20.5, 19.5, 18.3.

Aldehyde 12. A solution of methacrolein (0.5 ml, 5.2 mmol) and diene 11(0.1 g, 0.40 mmol) was stirred for 8 h at 25° C. under neat conditions.The excess methacrolein was then removed under reduced pressure. Thecrude product was subjected to chromatography (silica, 10-20% ether inhexanes) to afford aldehydes 12 and 12* (0.13 g, 0.40 mmol, 100%) as amixture of diastereomers (3:1-4:1 ratio at C13). 12 and 12*: colorlessoil; R_(f)=0.55 (silica, 25% ether in hexanes); 12: IR (film) ν_(max)3441, 2936, 1726, 1451, 1233, 1152; ¹H NMR (400 MHz, CDCl₃) δ9.70 (s,1H), 5.58 (m, 1H), 3.62 (s, 3H), 2.38-2.25 (m, 1H), 2.21-2.18 (m, 1H),2.17-1.98 (m, 4H), 1.96-1.62 (m, 6H), 1.61-1.58 (m, 1H), 1.57-1.43 (m,2H), 1.40-1.23 (m, 1H), 1.17 (s, 3H), 1.04 (s, 3H), 0.92 (s, 3H); ¹³C(100 MHz, CDCl₃) δ 207.6, 177.7, 148.3, 188.6, 51.3, 47.8, 47.0, 44.2,41.2, 39.3, 38.8, 38.1, 29.5, 28.4, 22.9, 22.5, 21.8, 20.6, 20.5, 19.7;12*: [α]₂₅ ^(D): +36.8 (c=0.7, C₆H₆); IR (film) ν_(max) 3441, 2936,1726, 1451, 1233, 1152; ¹H NMR (400 MHz, CDCl₃) δ9.64 (s, 1H), 5.42 (m,1H), 3.66 (s, 3H), 2.29-2.10 (m, 4H), 2.09-1.84 (m, 4H), 1.81-1.77 (m,2H), 1.75-1.63 (m, 2H), 1.62-1.58 (m, 2H), 1.57-1.45 (m, 1H), 1.43 (s,1H), 1.13 (s, 3H), 1.03 (s, 3H), 0.87 (s, 3H); ¹³C NMR (100 MHz, CDCl₃)δ 207.3, 177.5, 147.4, 114.6, 55.8, 51.3, 47.3, 44.5, 40.7, 40.4, 38.4,37.5, 31.5, 28.6, 25.0, 24.2, 21.9, 19.9, 19.6, 18.7.

The preferred way to purify the diastereomeric aldehydes is to reducethem with sodium borohydride in MeOH and separate the alcohols. Themajor compound (top diastereomer) can then be oxidized to the desiredaldehyde 12 upon treatment with Dess-Martin periodinane.

Alkene 13 (TTL3). A solution of (methyl)-triphenyl-phosphonium bromide(357 mg, 1.0 mmol) in THF (40 ml) was treated with 1M NaHMDS in THF(0.86 ml, 0.86 mmol). The resulting yellow mixture was allowed to stirat 25° C. for 30 min. After this time, a solution of aldehyde 12 (91 mg,0.29 mmol) in THF (10 ml) was added to the reaction via cannula. Thereaction mixture was stirred at 25° C. for 8 hours and then quenchedwith sodium bicarbonate (30 ml) and water (20 ml). The mixture waspoured into a separatory funnel containing ether (50 ml). Afterseparating the layers, the aqueous layer was extracted with ether (3×50ml). The organic layers were combined, dried with MgSO₄, condensed, andsubjected to chromatography (silica, 10% ether in hexanes) to affordalkene 13 (84 mg, 0.28 mmol, 97%). 13: colorless oil; R_(f)=0.75(silica, 25% ether in hexanes); 13: ¹H NMR (400 MHz, CDCl₃) δ 5.96dd,1H, J=16.8, 11.6 Hz), 5.50 (m, 1H), 4.98 (m, 2H), 3.62 (s, 3H),2.20-2.11 (m, 1H), 2.10-1.91 (m, 4H), 1.90-1.70 (m, 4H), 1.69-1.51 (m,3H), 1.50-1.38 (m, 3H), 1.36-1.24 (m, 1H), 1.17 (s, 3H), 1.04 (s, 3H),0.90 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ177.9, 149.1, 143.8, 117.9,111.7, 51.2, 47.7, 44.4, 41.4, 41.2, 38.9, 38.3, 37.7, 34.8, 30.4, 28.4,24.8, 23.1, 22.3, 22.2, 20.6, 19.8.

Acid 14 (TTL1). A solution of alkene 13 (84 mg, 0.28 mmol) in dimethylsulfoxide (20 ml) was treated with LiBr (121 mg, 1.4 mmol). The reactionmixture was refluxed at 180 C for 2 days. After cooling down, thereaction was diluted with water (30 ml) and extracted with ether (3×50ml). The organic layers were combined, dried with MgSO₄, concentrated,and subjected to chromatography (silica, 30% ether in hexanes) to affordcarboxylic acid 14 (TTL1) (78 mg, 0.26 mmol,). 14: white solid;R_(f)=0.30 (silica, 30% ether in hexanes); ¹H NMR (400 MHz, CDCl₃) δ5.96 (dd, 1H, J=14.4, 9.6 Hz), 5.52 (m, 1H), 4.98-4.95 (m, 2H),2.20-1.72 (m, 10H), 1.64-1.58 (m, 3H), 1.57-1.37 (m, 4H), 1.22 (s, 3H),1.04 (s, 3H), 0.99 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 182.9, 149.3,143.9, 118.1, 111.9, 47.5, 44.2, 41.3, 41.2, 38.9, 38.0, 37.6, 34.8,28.4, 24.7, 23.0, 22.4, 21.9, 20.3, 19.5.

Preparation of Ph₃P=¹⁴CH₂

Triphenylphosphine (0.16 g, 0.61 mmol) was added in a 15 ml reactionflask and dried overnight under vacuum at 25° C. To this flask was added2 ml of THF (dried and degassed under vacuum), followed by ¹⁴CH₃I (50mCi, 53 mCi/mmol, 0.9 mmol) dissolved in 1 ml of THF and the mixture wasstirred for 24 hours under argon. Potassium hexamethyldisilylamide (2.5ml, 1.25 mmol, 0.5 M in toluene) was then added and the reddish-yellowmixture was allowed to stirr for 3 h at 25° C.

Wittig Reaction with Ph₃P=¹⁴CH₂

The above mixture was cooled at −78° C. and treated with aldehyde 12 (63mg, 0.2 mmol) in dry THF (1.5 ml). The mixture was allowed to warmslowly to 25° C., stirred for 8 h and quenched with sodium bicarbonate(10 ml) and water (10 ml). The mixture was extracted with ether (3×50ml) and the organic layers were combined, dried with MgSO₄, condensed,and subjected to chromatography over silica gel (silica, 10% ether inhexanes) to afford alkene 13.

Alcohol 15. A solution of alkyne 9 (1.10 g, 4.2 mmol), thiophenol (1.37g, 12.4 mmol) and 2,2′-azobisisobutyronitrile (AIBN, 34.5 mg, 0.21 mmol)in xylene (25 ml) was stirred at 110° C. (under argon) for 18 h. Thereaction mixture was cooled to 25° C. and quenched with aqueoussaturated sodium bicarbonate (50 ml). The organic layer was extractedwith ethyl ether (3×50 ml), collected, dried (MgSO₄), concentrated andresidue was chromatographed (silica, 2-5% ethyl ether in hexane) toafford alchohol 15 (1.35 g, 3.6 mmol, 85.7%); 15: colorless liquid;R_(f)=0.51 (silica, 5% ethyl ether in hexanes); [α]²⁵D: +24.20 (c=1.0,benzene); IR (film) ν_(max) 2946.8, 1724.5, 1472.6, 1438.4, 1153.5,740.0, 690.9; ¹H NMR (500 MHz, CDCl₃) δ 7.20-7.60 (m, 5H), 5.23 (d, 1H,J=10.5 Hz), 5.12 (d, 1H, J=10.0 Hz), 3.62 (s, 3H), 2.08-2.24 (m, 2H),1.16-1.92 (m, 9H), 1.09 (s, 3H), 0.86-1.02 (m, 2H), 0.68 (s, 3H); ¹³CNMR (100 MHz, CDCl₃) δ 177.8, 151.7, 133.9, 133.7, 128.8, 127.9, 118.2,54.9, 53.5, 51.1, 44.3, 40.4, 38.1, 37.3, 28.7, 27.7, 25.5, 23.5, 19.5,18.5.

Diene 16. To a solution of alchohol 15 (1.10 g, 2.94 mmol) in hexamethylphosphoramide (HMPA, 10 ml) was added dropwise phosphorus oxychloride(0.50 g, 3.3 mmol) and the mixture was stirred at 25° C. until clear.Pyridine (0.26 ml, 3.23 mmol) was then added and the mixture was stirredat 150° C. (under argon) for 18 hrs. The reaction mixture was cooled to25° C. and quenched with aqueous saturated sodium bicarbonate (50 ml).The organic layer was extracted with ethyl ether (3×60 ml), collected,dried (MgSO₄) and concentrated and residue was chromatographed (silica,2-5% ethyl ether in hexane) to afford diene 16 (0.85 g, 2.38 mmol, 81%);16: colorless liquid; R_(f)=0.60 (silica, 5% ethyl ether in hexanes);[α]²⁵D: −17.30 (c=1.08, benzene); IR (film) ν_(max) 2957.0, 1726.6,1581.6, 1478.3, 1439.0, 1234.7, 1190.8, 1094.8, 1024.4, 739.1; ¹H NMR(500 MHz, CDCl₃) δ 7.20-7.60 (m, 5H), 6.43 (d, 1H, J=15.0 Hz), 6.36 (d,1H, J=14.5 Hz), 5.72 (m, 1H), 3.64 (s, 3H), 1.48-2.32 (m, 10H), 1.43 (s,3H), 1.21 (s, 3H), 1.05 (m,1H), 0.88 (s, 3H); ¹³C NMR (125 MHz, CDCl₃)δ177.9, 133.7, 129.1, 128.9, 128.6, 127.5, 126.2, 123.4, 120.9, 52.8,51.1, 43.7, 37.7, 37.3, 30.2, 28.3, 27.7, 20.1, 19.3, 18.3.

Aldehyde 17. To a solution of diene 16 (0.51 g, 1.43 mmol) andmethacrolein (0.30 g, 4.30 mmol) in dichloromethane (5 ml) at −20° C.was added under argon dropwise tin (IV) chloride (0.29 ml of 1M solutionin dichloromethane, 0.29 mmol). The resulting mixture was warmed to 0°C. within 1 hr and stirred at 0° C. for 18 h. The reaction was quenchedwith aqueous saturated sodium bicarbonate (15 ml) and the organic layerwas extracted with ethyl ether (3×20 ml). The combined organic layerswere dried (MgSO₄) and concentrated and residue was chromatographed(silica, 10-15% ethyl ether in hexane) to afford aldehyde 17 (0.51 g,1.19 mmol, 83.7%); 4: colorless liquid; R_(f)=0.48 (silica, 10% ethylether in hexanes); [α]²⁵D: +30.0 (c=1.13, benzene); IR (film) ν_(max)2930.8, 2871.4, 1724.9, 1458.4, 1226.4, 1149.8; ¹H NMR (500 MHz, CDCl₃)δ 9.51 (s, 1H), 7.20-7.60 (m, 5H), 5.57 (m, 1H), 3.65 (s, 3H), 1.20-2.32(m, 15H), 1.17 (s, 3H), 1.05 (s, 3H), 0.91 (s, 3H); ¹³C NMR (125 MHz,CDCl₃) δ203.6, 177.9, 153.7, 133.6, 133.5, 128.9, 127.8, 117.1, 51.3,49.1, 47.7, 44.2, 41.6, 38.7, 38.1, 31.2, 28.3, 27.8, 26.9, 21.7, 20.2,19.3, 18.6.

Alcohol 18. To a solution of aldehyde 17 (0.50 g, 1.17 mmol) inanhydrous ethanol (5 ml) was added portionwise sodium borohydride (50mg, 1.32 mmol) and the mixture was stirred for 30 min. Aqueous saturatedsodium bicarbonate (10 ml) was then added and the mixture was extractedwith ethyl ether (3×20 ml). The organic layer was collected, dried(MgSO₄) and concentrated. The residue was redissolved in tetrahydrofuran(5 ml) and treated with excess of Raney Nickel under argon at 65° C. for10 min. The reaction mixture was filtered, and the filtrate was dried(MgSO₄) and concentrated, and the residue was chromatographed (silica,2-5% ethyl ether in hexane) to afford alcohol 18 as a major compound(0.21 g, 0.65 mmol, overall yield 56.1%. Note: the overall yield for theabove two reactions is 91%); 18: colorless liquid; R_(f)=0.39 (silica,30% ethyl ether in hexanes); [α]²⁵D: −6.70 (c=1.0, benzene); IR (film)ν_(max) 3436.8, 2929.0, 2872.2, 1728.1, 1433.9, 1260.6, 1029.7, 801.6;¹H NMR (500 MHz, CDCl₃) δ 5.37 (m, 1H), 3.62 (s, 3H), 2.28 (bs, 1H),2.06-2.20 (m, 2H), 1.20-2.00 (m, 12H), 1.16 (s, 3H), 0.99 (m, 1H), 0.86(s, 3H), 0.84 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ178.2, 150.4, 116.4,73.6, 51.2, 47.9, 44.2, 41.9, 38.8, 38.2, 34.3, 33.9, 28.3, 28.2, 27.8,22.1, 20.3, 20.1, 18.9.

Alkene 19. To a solution of alchohol 18 (20.0 mg, 0.062 mmol) indichloromethane (2 ml) was added Dess-Martin periodinane (35 mg, 0.08mmol) in portions, and the mixture was stirred at 25° C. for 30 min. Thereaction was quenched with aqueous saturated sodium bicarbonate (5 ml)and extracted with ethyl ether (3×10 ml). The organic layer wascollected, dried (MgSO₄) and concentrated. The residue was redissolvedin tetrahydrofuran (0.5 ml) and added under argon to a yellow suspensionof (methyl)triphenyl-phosphonium bromide (60 mg, 0.17 mmol) and sodiumbis(trimethylsilyl)amide (0.14 ml of 1.0 M in THF) in THF (1.5 ml).After stirring at 25° C. for 18 h the mixture was diluted with aqueoussaturated sodium bicarbonate (5 ml) and extracted with ethyl ether (3×10ml). The organic layer was collected, dried (MgSO₄), concentrated andresidue was chromatographed (silica, 2-5% ethyl ether in hexane) toafford alkene 19 (16.8 mg, 0.05 mmol, the overall yield for the two-stepreactions is 86%); 19: colorless liquid; R_(f)=0.74 (silica, 5% ethylether in hexanes); [α]²⁵D: −14.40 (c=0.50, benzene); IR (film) ν_(max)2929.5, 2873.4, 1726.8, 1637.7, 1460.7, 1376.8, 1225.1, 1150.4, 997.8,908.7; ¹H NMR (500 MHz, CDCl₃) δ 5.82 (dd, 1H), 5.39 (m, 1H), 4.85-4.94(dd, 2H), 3.64 (s, 3H), 2.30 (bs, 1H), 2.14 (m, 1H), 2.02 (m, 1H),1.80-1.98 (m, 2H), 1.68-1.80 (m, 2H), 1.20-1.68 (m, 7H), 1.18 (s, 3H),0.96-1.08 (m, 2H), 0.95 (s, 3H), 0.88 (s, 3H); ¹³C NMR (125 MHz, CDCl₃)δ178.3, 150.4, 125.6, 116.6, 109.2, 51.2, 47.9, 44.3, 41.9, 41.8, 38.3,38.2, 37.4, 34.8, 30.2, 29.6, 28.6, 28.4, 27.8, 22.1, 20.4, 19.0

.

Compound of Formula (I). To a solution of alkene 19 (16.8 mg, 0.05 mmol)in N,N-dimethylformamide (2 ml) added lithium bromide (5.0 mg, 0.06mmol) and the mixture was refluxed at 190° C. for 1 hr. The reactionmixture was then cooled to 25° C., diluted with H₂O (5 ml) and extractedwith ethyl acetate (3×10 ml). The organic layer was collected, dried(MgSO₄) and concentrated and residue was chromatographed (silica, 15-20%ethyl ether in hexane) to afford Formula (I) (14.9 mg, 0.05 mmol,92.6%);

Compound of Formula (I) is a colorless liquid; R_(f)=0.20 (silica, 30%ethyl ether in hexanes); [α]²⁵D: −6.0 (c=0.33, benzene); IR (film)ν_(max) 3080.6, 2928.9, 2857.6, 1693.6, 1638.2, 1464.7, 1413.8, 1376.4,1263.1, 1179.3, 1095.9, 1027.5, 999.2, 909.2, 801.7; ¹H NMR (500 MHz,CDCl₃) δ 5.82 (dd, 1H), 5.40 (m, 1H), 4.85-4.95 (dd, 2H), 2.30 (bs, 1H),2.16 (m, 1H), 2.02 (m, 1H), 1.80-1.98 (m, 2H), 1.70-1.84 (m, 2H),1.10-1.70 (m, 7H), 1.24 (s, 3H), 1.00-1.10 (m, 2H), 0.99 (s, 3H), 0.95(s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 150.3, 149.9, 116.7, 109.2, 47.9,41.8, 41.7, 38.3, 38.2, 37.4, 34.8, 31.8, 28.6, 28.5, 27.7, 22.6, 22.4,22.1, 20.3, 18.9.

Methods of Using the Invention

The in vitro and in vivo methods described above as part of the presentinvention also establish the selectivity of a TNF-α or IL-1 modulator.It is recognized that chemicals can modulate a wide variety ofbiological processes or be selective. Panels of cells based on thepresent invention can be used to determine the specificity of thecandidate modulator. Selectivity is evident, for example, in the fieldof chemotherapy, where the selectivity of a chemical to be toxic towardscancerous cells, but not towards non-cancerous cells, is obviouslydesirable. Selective modulators are preferable because they have fewerside effects in the clinical setting. The selectivity of a candidatemodulator can be established in vitro by testing the toxicity and effectof a candidate modulator on a plurality of cell lines that exhibit avariety of cellular pathways and sensitivities. The data obtained fromthese in vitro toxicity studies may be extended to animal models,including accepted animal model studies and human clinical trials, todetermine toxicity, efficacy, and selectivity of the candidatemodulator.

The present invention also encompasses the compositions, produced by themethods of the invention, in pharmaceutical compositions comprising apharmaceutically acceptable carrier prepared for storage and subsequentadministration, which have a pharmaceutically effective amount of theproducts disclosed above in a pharmaceutically acceptable carrier ordiluent. Acceptable carriers or diluents for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). Preservatives, stabilizers, dyes and even flavoring agentsmay be provided in the pharmaceutical composition. For example, sodiumbenzoate, ascorbic acid and esters of p-hydroxybenzoic acid may be addedas preservatives. In addition, antioxidants and suspending agents may beused.

These TNF-α or IL-1 modulator compositions may be formulated and used astablets, capsules, or elixirs for oral administration; suppositories forrectal administration; sterile solutions, suspensions for injectableadministration; patches for transdermal administration, and sub-dermaldeposits and the like. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations (forexample, liposomes), may be utilized.

The pharmaceutically effective amount of the TNF-α or IL-1 modulatorcomposition required as a dose will depend on the route ofadministration, the type of animal being treated, and the physicalcharacteristics of the specific animal under consideration. The dose canbe tailored to achieve a desired effect, but will depend on such factorsas weight, diet, concurrent medication and other factors which thoseskilled in the medical arts will recognize.

In practicing the methods of the invention, the products or compositionscan be used alone or in combination with one another, or in combinationwith other therapeutic or diagnostic agents. These products can beutilized in vivo, ordinarily in a mammal, preferably in a human, or invitro. In employing them in vivo, the products or compositions can beadministered to the mammal in a variety of ways, including parenterally,intravenously, subcutaneously, intramuscularly, colonically, rectally,vaginally, nasally or intraperitoneally, employing a variety of dosageforms. Such methods may also be applied to testing chemical activity invivo.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art using routine pharmacologicalmethods. Typically, human clinical applications of products arecommenced at lower dosage levels, with dosage level being increaseduntil the desired effect is achieved. Alternatively, acceptable in vitrostudies can be used to establish useful doses and routes ofadministration of the compositions identified by the present methodsusing established pharmacological methods.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage for the products of the present invention can range broadlydepending upon the desired affects and the therapeutic indication.Typically, dosages may be between about 10 microgram/kg and 100 mg/kgbody weight, preferably between about 100 microgram/kg and 10 mg/kg bodyweight. Alternatively dosages may be based and calculated upon thesurface area of the patient, as understood by those of skill in the art.Administration is preferably oral on a daily or twice daily basis.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. See forexample, Fingl et al., in The Pharmacological Basis of Therapeutics,1975. It should be noted that the attending physician would know how toand when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministrated dose in the management of the disorder of interest willvary with the severity of the condition to be treated and to the routeof administration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. A variety oftechniques for formulation and administration may be found inRemington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,Easton, Pa. (1990). Suitable administration routes may include oral,rectal, transdermal, vaginal, transmucosal, or intestinaladministration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. Use of pharmaceutically acceptable carriersto formulate the compounds herein disclosed for the practice of theinvention into dosages suitable for systemic administration is withinthe scope of the invention. With proper choice of carrier and suitablemanufacturing practice, the compositions of the present invention, inparticular, those formulated as solutions, may be administeredparenterally, such as by intravenous injection. The compounds can beformulated readily using pharmaceutically acceptable carriers well knownin the art into dosages suitable for oral administration. Such carriersenable the compounds of the invention to be formulated as tablets,pills, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. All molecules present in an aqueoussolution at the time of liposome formation are incorporated into theaqueous interior. The liposomal contents are both protected from theexternal micro-environment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm.Additionally, due to their hydrophobicity, small organic molecules maybe directly administered intracellularly.

Pharmaceutical compositions suitable for use as herein described includecompositions wherein the TNF-α or IL-1 modulators are contained in aneffective amount to achieve the TNF-α or IL-1 modulatory purpose.Determination of the effective amounts is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein. In addition to the active ingredients, thesepharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. The preparations formulated for oraladministration may be in the form of tablets, dragees, capsules, orsolutions. The pharmaceutical compositions of the present invention maybe manufactured in a manner that is itself known, for example, by meansof conventional mixing, dissolving, granulating, dragee-making,levitating, emulsifying, encapsulating, entrapping, or lyophilizingprocesses.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or other organic oilssuch as soybean, grapefruit or almond oils, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents that increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. For this purpose, concentratedsugar solutions may be used, which may optionally contain gum arabic,talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. Such formulations can be madeusing methods known in the art (see, for example, U.S. Pat. No.5,733,888 (injectable compositions); U.S. Pat. No. 5,726,181 (poorlywater soluble compounds); U.S. Pat. No. 5,707,641 (therapeuticallyactive proteins or peptides); U.S. Pat. No. 5,667,809 (lipophilicagents); U.S. Pat. No. 5,576,012 (solubilizing polymeric agents); U.S.Pat. No. 5,707,615 (anti-viral formulations); U.S. Pat. No. 5,683,676(particulate medicaments); U.S. Pat. No. 5,654,286 (topicalformulations); U.S. Pat. No. 5,688,529 (oral suspensions); U.S. Pat. No.5,445,829 (extended release formulations); U.S. Pat. No. 5,653,987(liquid formulations); U.S. Pat. No. 5,641,515 (controlled releaseformulations) and U.S. Pat. No. 5,601,845 (spheroid formulations).

Compounds of the present invention can be evaluated for efficacy andtoxicity using known methods. For example, the toxicology of aparticular compound of the present invention, or of a subset of thecompounds of the present invention sharing certain chemical moieties,can be established by determining in vitro toxicity towards a cell line,such as a mammalian, and preferably human, cell line. The results ofsuch studies are often predictive of toxicity in animals, such asmammals, or more specifically, humans. Alternatively, the toxicity ofparticular compounds of the present invention in an animal model, suchas mice, rats, rabbits, or monkeys, may be determined using knownmethods. The efficacy of a particular compound of the present inventionmay be established using several art recognized methods, such as invitro methods, animal models, or human clinical trials. Art-recognizedin vitro models exist for nearly every class of condition, including theconditions abated by the present invention, including cancer,cardiovascular disease, and various immune disfunction. Similarly,acceptable animal models may be used to establish efficacy of chemicalsto treat such conditions. When selecting a model to determine efficacy,the skilled artisan can be guided by the state of the art to choose anappropriate model, dose, and route of administration, and regime. Ofcourse, human clinical trials can also be used to determine the efficacyof a compound of the present invention in humans.

When used as an anti-inflammatory agent, an anti-cancer agent, atumor-growth-inhibiting compound, or as a means of treatingcardiovascular disease, the compounds of Formulae (II), (IIA), andpreferably (IIB) can be administered by either oral or a non-oralpathways. When administered orally, it can be administered in capsule,tablet, granule, spray, syrup, or other such form. When administerednon-orally, it can be administered as an aqueous suspension, an oilypreparation or the like or as a drip, suppository, salve, ointment orthe like, when administered via injection, subcutaneously,intreperitoneally, intravenously, intramuscularly, intradermally, or thelike. Similarly, it may be administered topically, rectally, orvaginally, as deemed appropriate by those of skill in the art forbringing the compound of the invention into optimal contact with atumor, thus inhibiting the growth of the tumor. Local administration atthe site of the tumor or other disease condition is also contemplated,either before or after tumor resection, or as part of an art-recognizedtreatment of the disease condition. Controlled release formulations,depot formulations, and infusion pump delivery are similarlycontemplated.

The compounds of Formulae (II) and (IIA), and preferably (IIB), whenused as an antitumor agent or as a treatment for any otherabove-identified disease condition, may be orally or non-orallyadministered to a human patient in the amount of about 0.0007 mg/day toabout 7,000 mg/day of the active ingredient, and more preferably about0.07 mg/day to about 70 mg/day of the active ingredient at, preferably,one time per day or, less preferably, over two to about ten times perday. Alternatively and also preferably, the compound of the inventionmay preferably be administered in the stated amounts continuously by,for example, an intravenous drip. Thus, for a patient weighing 70kilograms, the preferred daily dose of the active anti-tumor ingredientwould be about 0.0007 mg/kg/day to about 35 mg/kg/day, and morepreferable, 0.007 mg/kg/day to about 0.035 mg/kg/day. Nonetheless, aswill be understood by those of skill in the art, in certain situationsit may be necessary to administer the anti-tumor compound of theinvention in amounts that excess, or even far exceed, the above-stated,preferred dosage range to effectively and aggressively treatparticularly advanced or lethal tumors.

To formulate the compound of Formula (II), the compound of Formula(IIA), or the compound of Formula (IIB), as a tumor-growth-inhibiting oranti-viral compound, known surface active agents, excipients, smoothingagents, suspension agents and pharmaceutically acceptable film-formingsubstances and coating assistants, and the like may be used. Preferablyalcohols, esters, sulfated aliphatic alcohols, and the like may be usedas surface active agents; sucrose, glucose, lactose, starch,crystallized cellulose, mannitol, light anhydrous silicate, magnesiumaluminate, magnesium methasilicate aluminate, synthetic aluminumsilicate, calcium carbonate, sodium acid carbonate, calcium hydrogenphosphate, calcium carboxymethyl cellulose, and the like may be used asexcipients; magnesium stearate, talc, hardened oil and the like may beused as smoothing agents; coconut oil, olive oil, sesame oil, peanutoil, soya may be used as suspension agents or lubricants; celluloseacetate phthalate as a derivative of a carbohydrate such as cellulose orsugar, or methyiacetate-methacrylate copolymer as a derivative ofpolyvinyl may be used as suspension agents; and plasticizers such asester phthalates and the like may be used as suspension agents. Inaddition to the foregoing preferred ingredients, sweeteners, fragrances,colorants, preservatives and the like may be added to the administeredformulation of the compound of the invention, particularly when thecompound is to be administered orally.

In the case of using the compound of Formula (II), Formula (IIA), and/orFormula (IIB) as a means of treating skin redness, the compound mayalternatively be administered topically as a salve or ointment, inconjunction with a pharmaceutically acceptable carrier.

In the case of using the compound of Formula (II), Formula (IIA), and orFormula (IIB) as a biochemical test reagent, as described above, thecompound of the invention may be dissolved in an organic solvent orhydrous organic solvent and directly applied to any of various culturedcell systems. Usable organic solvents include, for example, methanol,methylsulfoxide, and the like. The formulation can, for example, be apowder, granular or other solid inhibitor, or a liquid inhibitorprepared using an organic solvent or a hydrous organic solvent. While apreferred concentration of the compound of the invention for use as acell cycle inhibitor is generally in the range of about 1 to about 100μg/ml, the most appropriate use amount varies depending on the type ofcultured cell system and the purpose of use, as will be appreciated bypersons of ordinary skill in the art. Also, in certain applications itmay be necessary or preferred to persons of ordinary skill in the art touse an amount outside the foregoing range.

The present invention also encompasses the compositions of Formula (II),Formula (IIA), and/or Formula (IIB) in a pharmaceutical compositionscomprising a pharmaceutically acceptable carrier. Such compositions maybe prepared for storage and for subsequent administration. Acceptablecarriers or diluents for therapeutic use are well known in thepharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).For example, such compositions may be formulated and used as tablets,capsules or solutions for oral administration; suppositories for rectalor vaginal administration; sterile solutions or suspensions forinjectable administration. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Suitable excipients include, but are not limited to, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations (forexample, liposomes), may be utilized.

The pharmaceutically effective amount of the composition required as adose will depend on the route of administration, the type of animalbeing treated, and the physical characteristics of the specific animalunder consideration. The dose can be tailored to achieve a desiredeffect, but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill recognize.

The products or compositions of the invention, as described above, maybe used alone or in combination with one another, or in combination withother therapeutic or diagnostic agents. These products can be utilizedin vivo or in vitro. The useful dosages and the most useful modes ofadministration will vary depending upon the age, weight and animaltreated, the particular compounds employed, and the specific use forwhich these composition or compositions are employed. The magnitude of adose in the management or treatment for a particular disorder will varywith the severity of the condition to be treated and to the route ofadministration, and depending on, the disease conditions and theirseverity, the compositions of the present invention may be formulatedand administered either systemically or locally. A variety of techniquesfor formulation and administration may be found in Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa.(1990).

Various references, publications, and patents are cited herein. To theextent permitted by law, each of these references, publications, andpatents is hereby incorporated by reference herein in its entirety.

EXAMPLES

The following examples are meant to illustrate specific, preferredembodiments of the invention, and are not meant to limit the scope ofprotection afforded by the invention. The following examples,specifically Example 1-8, demonstrate that representative compounds ofthe classes of compounds described herein have been synthesized.Examples 9-17 exhibit, in mammalian cells which present an acceptablepreliminary model for human efficacy and safety, treated with increasingdoses of the compound of Formula (I), as synthesized in Example 1, andcompounds of Formula (IIB), as herein designated TTL1 through TTL4, assynthesized in accordance with the processes of Example 1, and, moreparticularly, as in Examples 2-5, at concentrations as high as 10 μg/mlshowed similar viability compared to untreated controls indicating thatthe inhibitory effects of the evaluated compounds on TNF-α synthesiswere not mediated by a direct cytotoxic effect.

Subsequent studies with certain preferred compounds of the inventiondemonstrated that TTL1 exhibited approximately ten (10) fold greateractivity compared to THE SYNTHETIC COMPOUND OF FORMULA (I) in inhibitingTNF-α and IL-1 synthesis. TTL3 which contains an additional chemicalmodification exhibited approximately 100 times greater activity thanTTL1. It is important to note that similar to the compound of Formula(I), neither TTL1 nor TTL3 significantly inhibited IL-6 synthesis.

Example 1 Stereoselective Synthesis of Compounds of Formulae (I) and(II)

The first stereoselective synthesis of Compound of Formula (I) has beenaccomplished. Our synthetic plan departs from (−) Wieland-Miesher ketone(107), see FIG. 18, and calls upon a Diels-Alder cycloaddition reactionfor the construction of the C ring of 101. The described synthesisconfirms the proposed stereochemistry of 101 and represents an efficiententry into an unexplored class of biologically active diterpenes.

The root bark of Acanthopanax koreanum Nakai (Araliaceae), a deciduousshrub that grows in The Republic of Korea, has been used traditionallyas a tonic, sedative, and as a remedy for rheumatism and diabetes.(Medicinal Plants of East and Southeast Asia, Perry, L. M.; Metzger, J.Eds.; MIT Press, Cambridge, Mass. and London, 1980). In their study ofthe pharmacologically active extracts of this folk medicine, Chung andco-workers have isolated and structurally characterized a novelditerpene, that was subsequently named acanthoic acid (101). ((a) Kim,Y.-H.; Chung, B. S.; Sankawa, U. J. Nat. Prod. 1988, 51, 1080-1083; (b)Kang, H.-S.; Kim, Y.-H.; Lee, C.-S.; Lee, J.-J.; Choi, I.; Pyun, K.-H.Cellular Immunol. 1996, 170, 212-221; (c) Kang, H.-S.; Song, H. K.; Lee,J.-J.; Pyun, K.-H.; Choi, I. Mediators Inflamm. 1998, 7, 257-259).

From the biosynthesis standpoint, 101 belongs to a rather large familyof pimaradiene diterpenes, which may be best represented by pimaric acid(102). (Ruzicka, L.; Sternbach, L.; J. Am. Chem. Soc. 1948, 70,2081-2085; Ireland, R. E.; Schiess, P. W. Tetrahedron Lett. 1960, 25,37-43; Wenkert, E.; Buckwalter, B. L. J. Am. Chem. Soc. 1972, 94,4367-4372; Wenkert, E.; Chamberlin, J. W. J. Am. Chem. Soc. 1959, 81,688-693). The structure of the compound of Formula (I) is distinguishedby an uncommon connectivity across the rigid tricyclic core, which maybe held accountable for its pharmacological profile. Indeed, the recentisolation of this compound has allowed studies into its biologicalactivity and verified its medicinal potential. (Kang, H.-S.; Kim, Y.-H.;Lee, C.-S.; Lee, J.-J.; Choi, I.; Pyun, K.-H. Cellular Immunol. 1996,170, 212-221; Kang, H.-S.; Song, H. K.; Lee, J.-J.; Pyun, K.-H.; Choi,I. Mediators Inflamm. 1998, 7, 257-259)). More specifically, acanthoicacid was found to exhibit promising anti-inflammatory and antifibroticactivities that presumably arise by inhibiting the production of thepro-inflammatory cytokines: tumor necrosis factor-alpha (TNF-α) andinterleukin-1 (IL-1). See Tumor Necrosis Factors. The Molecules andtheir Emerging Role in Medicine, B. Beutler, Ed.; Raven Press, N.Y.1992; Aggarwal, B.; Puri, R. Human Cytokines: Their Role in Disease andTherapy; Blackwell Science, Inc.: U.S.A., 1995; Thorpe, R.; Mire-Sluis,A. Cytokines; Academic Press: San Diego, 1998; Kurzrock, R.; Talpaz, M.Cytokines: Interleukins and Their Receptors; Kluwer Academic Publishers:U.S.A., 1995; Szekanecz, Z.; Kosh, A. E.; Kunkel, S. L.; Strieter, R. M.Clinical Pharmacol. 1998, 12, 377-390; Camussi, G.; Lupin, E. Drugs1998, 55, 613-620; Newton, R. C.; Decicco, C. P. J. Med. Chem. 1999, 42,2295-2314.

This inhibition was concentration dependent and cytokine-specific sinceunder the same conditions the production of IL-6 or IFN-γ(interferon-gamma) were not affected. In addition, acanthoic acid wasfound to be active upon oral administration and showed minimal toxicityin experiments performed in mice and rats.

The combination of uncommon structure and promising pharmacologicalactivity displayed by 101 prompted us to extend our synthetic studies,see Xiang, A. X.; Watson, D. A.; Ling. T.; Theodorakis, E. A. J. Org.Chem. 1998, 63, 6774-6775; Ling, T.; Xiang, A. X.; Theodorakis, E. A.Angew. Chem. Int. Ed. Engl. 1999, 38, 3089-3091, to this family ofbiologically important metabolites. This example provides astereoselective total synthesis of (−) acanthoic acid and the compoundsof Formula (II) and, as shown in Examples 2-6, provides the basis forthe total synthesis of the compounds of Formula (IIB). This Example alsoconfirms the structure and absolute stereochemistry of 101.

The retrosynthetic strategy towards acanthoic acid is illustrated inFIG. 20. The C ring of 101 is envisioned to be constructed by aDiels-Alder cycloaddition reaction, thereby revealing dienophile 103 andan appropriately substituted diene, such as 104, as ideal couplingpartners. See Oppolzer, W in Comprehensive Org. Synthesis, Trost, B. M.Ed.; Oxford, N.Y.; Pergamon Press, 1991, 315-399. This reactionintroduces both the unsaturation at the C9-C11 bond and the desiredstereochemistry at the C8 and C13 carbons, permitting a convenientbranch point between the syntheses of the compounds of Formula (II) andthe compounds of Formula (IIB). Diene 104 could be produced byfunctionalization of ketone 105, whose C4 quaternary center wasprojected to be formed by a stereocontrolled alkylation of β-ketoester107. This analysis suggested the use of (−) Wieland-Miesher ketone 107as a putative starting material. Application of such a plan to thesynthesis of acanthoic acid is depicted in FIGS. 21 and 23, as Schemes 5and 6. All compounds exhibited satisfactory spectral and analyticaldata.

The synthesis began with optically pure enone 107, which was readilyavailable through a D-proline-mediated asymmetric Robinson annulation(75-80% yield, >95% ee). See Buchschacher, P.; Fuerst, A.; Gutzwiller,J. Org. Synth. Coll. Vol. VII 1990, 368-3372.). Selective ketalizationof the C9 ketone group of 107, followed by reductive alkylation acrossthe enone functionality with methyl cyanoformate afforded ketoester 106in 50% overall yield. See Crabtree, S. R.; Mander, L. N.; Sethi, P. S.Org. Synth. 1992, 70, 256-263. To introduce the desiredfunctionalization at the C4 position, a second reductive alkylationprocedure was implemented, see Coates, R. M.; Shaw, J. E. J. Org. Chem.1970, 35, 2597-2601; Coates, R. M.; Shaw, J. E. J. Org. Chem. 1970, 35,2601-2605. Compound 106 was first transformed to the correspondingmethoxymethyl ether 108, which upon treatment with lithium in liquidammonia and iodomethane gave rise to ester 110 in 58% overall yield andas a single diastereomer. See Welch, S. C.; Hagan, C. P. Synthetic Comm.1973, 3, 29-32; Welch, S. C.; Hagan, C. P.; Kim, J. H.; Chu, P. S. J.Org. Chem. 1977, 42, 2879-2887; Welch, S. C.; Hagan, C. P.; White, D.H.; Fleming, W. P.; Trotter, J. W. J. Amer. Chem. Soc. 1977, 99,549-556. The stereoselectivity of this addition arose from the strongpreference of the intermediate enolate 109 to undergo alkylation at theless hindered equatorial side.

With the bicyclic core at hand, the C ring was constructed. The C-ringwas formed via a Diels-Alder reaction between methacrolein 103, see forexample, FIG. 21, and the sulfur-containing diene 104. The synthesis of104 was initiated with an acid-catalyzed deprotection of the C9 ketal of110, followed by alkylation of the resulting ketone 105 with lithiumacetylide.ethylene diamine complex. See Das, J.; Dickinson, R. A.;Kakushima, M.; Kingston, G. M.; Reid, G. R.; Sato, Y.; Valenta, Z. Can.J. Chem. 1984, 62, 1103-1111). This sequence afforded alkyne 111 as an8:1 diasteromeric mixture at C9 (in favor of the isomer shown) and in86% overall yield. At this point, the diastereofacial selectivity of theDiels-Alder reaction was evaluated, as was the overall feasibility ofusing a non-functionalized diene, such as 112. To this end, thediastereomeric mixture of propargyl alcohols 111 was partially reduced(H₂, Lindlar's catalyst) and dehydrated (BF₃.Et₂O) to produce diene 112in 90% yield. (Coisne, J.-M.; Pecher, J.; Declercq, J.-P.; Germain, G.;van Meerssche, M. Bull. Soc. Chim. Belg. 1980, 89, 551-557). TheDiels-Alder cycloaddition between 112 and methacrolein (103) under neatconditions at 25° C., afforded in quantitative yield a mixture of twodiastereomeric aldehydes that were separated after reduction with sodiumborohydride. The resulting alcohols 114 and 115 were transformed to thecorresponding p-bromobenzoate esters (compounds 116 and 117respectively), which upon recrystallization with dichloromethane/ethanolyielded crystals suitable for X-Ray analysis (FIG. 22).

The results of the X-ray analyses established that the tricyclic systemhad the expected stereochemistry at the C4 position and confirmed thatthe Diels-Alder reaction proceeded with exclusive endo orientation.Methacrolein was shown to produce exo Diels Alder products when reactingwith cyclopentadiene: Kobuke, Y.; Fueno, T.; Furukawa, J. J. Am. Chem.Soc. 1970, 92, 6548-6553. This surprising observation was rationalizedbased on the steric repulsion exhibited by the methyl group: Yoon, T.;Danishefsky, S. J.; de Gala, S. Angew. Chem. Int. Ed. Engl. 1994, 33,853-855). Second, after reduction, the major product of thecycloaddition was shown to be alcohol 114, which had the desiredstereochemistry at the C8 center, thereby demonstrating a strongpreference of diene 112 to undergo reaction with 103, see for example,FIG. 21, from the α-face (bottom side attack). Moreover, these dataindicated that synthesis of acanthoic acid would require an inversion inthe orientation of the incoming dienophile.

As discussed in Example 2-8, below, absent inversion of the incomingdienophile, the wholly novel compounds of Formula (IIB) weresynthesized. The choice of an appropriate substituted dienophile allowsessentially limitless selection of the R₁, and R₁₂ groups of thecompounds of Formula (IIB).

The inversion of the dienophile required for the synthesis of thecompound of Formula (I), its naturally occurring analogs, and thecompounds of Formula (II) and (IIA), was accomplished by altering theatomic orbital coefficients at the termini of the diene, supporting theuse of a heteroatom-containing diene, such as 104, during thecycloaddition. See generally Overman, L. E.; Petty, C. B.; Ban, T.;Huang, G. T. J. Am. Chem. Soc. 1983, 105, 6335-6338; Trost, B. M.;Ippen, J.; Vladuchick, W. C. J. Am. Chem. Soc. 1977, 99, 8116-8118;Cohen, T.; Kozarych, Z. J. Org. Chem. 1982, 47, 4008-4010; Hopkins, P.B.; Fuchs, P. L. J. Org. Chem. 1978, 43, 1208-1217; Petrzilka, M.;Grayson, J. I. Synthesis, 1981, 753-786). The construction of diene 104and its utilization for the synthesis of 101 is shown in FIG. 23, Scheme6.

Compound 104 was produced by a radical addition of thiophenol ontoalkyne 111 (Greengrass, C. W.; Hughman, J. A.; Parsons, P. J. J. Chem.Soc. Chem. Commun. 1985, 889-890), followed by POCl₃-mediateddehydration of the resulting allylic alcohol (Trost, B. M.; Jungheim, L.N. J. Am. Chem. Soc. 1980, 102, 7910-7925; Mehta, G.; Murthy, A. N.;Reddy, D. S.; Reddy, A. V. J. Am. Chem. Soc. 1986, 108, 3443-3452) (2steps, 70% yield). Interestingly, this dehydration was also attemptedwith BF₃.Et₂O, but proved ineffective in this case. With a substantialamount of 104 at hand, we investigated the Diels-Alder reaction, using103 as the dienophile. Several thermal- (−78 to 80° C.) and Lewisacid-(BF₃.Et₂O, TiCl₄, AlCl₃ and SnCl₄) catalyzed Diels-Alder conditionswere tested. Best results were obtained with SnCl₄ in methylene chlorideat −20° C. and afforded aldehyde 118 in 84% yield as a 4.2:1 mixture ofdiastereomers. To simplify the product characterization and allowadequate separation, this mixture was reduced with NaBH4 and reductivelydesulfurized using Raney Ni. Alcohols 119 and 120 were thus obtained in91% overall yield. The structure of these compounds was assigned bycomparison to the products isolated from the reaction between 103 and112. Treatment of the major diastereomer 120 with Dess-Martinperiodinane, followed by Wittig methylenation installed the alkenefunctionality at the C13 center and produced 121 in 86% overall yield.The C-19 carboxylic acid was then deprotected. Exposure of 121 to LiBrin refluxing DMF gave rise to acanthoic acid 101 in 93% yield via anS_(N) ²-type displacement of the acyloxyl functionality. See Bennet, C.R.; Cambie, R. C. Tetrahedron 1967, 23, 927-941. Synthetic 101 hadidentical spectroscopic and analytical data with those reported for thenatural product.

This Example provides a concise, stereoselective synthesis of Compound101. The synthetic strategy is highlighted by the implementation of aDiels-Alder reaction between diene 104 and methacrolein (103), which setthe stereochemistry at the C13 and C8 carbon centers. The describedsynthesis of 101 requires fourteen steps (starting with enone 107) andproceeds in approximately 9% overall yield. The overall efficiency andversatility of our strategy sets the foundation for the preparation ofdesigned analogs with improved pharmacological profiles.

Examples 2-8 Stereoselective Synthesis of Compounds of Formula (IIB)

The procedure outline in Example 1, and depicted in FIG. 23, Scheme 6,may be modified or truncated to yield the compounds of Formula (II) orFormula (IIB).

Example 2

The compound herein designated TTL4 was synthesized by following theprocedures of Example 1, as depicted in FIG. 21, to yield compound 114,herein designated TTL4.

Example 3

The compound herein designated TTL2 was synthesized by following theprocedures of Example 1, as depicted in FIG. 21, to yield compound 114.Similar to the reaction depicted in FIG. 23, step (h), the compound 114was then reacted with 3.0 equivalents LiBr, in DMF, at 160° C., forapproximately three hours, to a yield of approximately 93% of thecompound herein designated TTL2.

Example 4

The compound herein designated TTL3 was synthesized by following theprocedures as depicted in FIG. 14, to yield compound 13. This compoundis herein designated TTL3.

Example 5

The compound herein designated TTL1 was synthesized by following theprocedures as depicted in FIG. 14, to yield compound 13. This compoundis herein designated TTL1.

Example 6

A compound of Formulae (IIB) wherein R₁₅ is a hydrogen and R₉ and R₁₅are separately selected from the group consisting of C₁-C₆ alkyl, andC₁-C₆ substituted alkyl is synthesized by following the procedurestherefor of Example 1, except that the dienophile is selected from oneof the compound of Formulae (III) wherein R₁₅ is a hydrogen and R₉ andR₁₅ are separately selected from the group consisting of C₁-C₆ alkyl,and C₁-C₆ substituted alkyl, as in this Example.

Example 7

Specifically, a compound of Formulae (IIB) wherein R₁₄ is a hydrogen andR₉ and R₁₅ are separately selected from the group consisting of C₁-C₆alkyl, and C₁-C₆ substituted alkyl is synthesized by following theprocedures therefor of Example 1, except that the dienophile is selectedfrom one of the compound of Formulae (III) wherein R₁₄ is a hydrogen andR₉ and R₁₅ are separately selected from the group consisting of C₁-C₆alkyl, and C₁-C₆ substituted alkyl, as in this Example.

Example 8

A compound of Formulae (IIB) wherein R₁₄ is a hydrogen, and R₉ and R₁₅are separately selected from C₂-C₆ alkenyl, C₂-C₆ substituted alkenyl,C₁-C₆ alcohol, and C₅-C₆ aryl, is synthesized by following theprocedures therefor of Example 1, except that the dienophile is selectedfrom one of the compound of Formulae (III) wherein R₁₄ is a hydrogen andR₉ and R₁₅ are separately selected from C₂-C₆ alkenyl, C₂-C₆ substitutedalkenyl, C₁-C₆ alcohol, and C₅-C₆ aryl, as in this Example.

Examples 9-17

Materials and Methods.

Murine macrophage cells RAW 264.7 (1×10⁶/ml) were pretreated for 30-60minutes with varying doses of the synthetic compound of Formula (I), thesynthetic compound of formula (I) and a panel of analogs (diluted in0.5% DMSO) prior to stimulation with various agents such aslipopolysaccharide (LPS) or a gram positive agent like heat-killed Staphaureus (SAC). Supernatants collected over a 72-hour period will beassayed for the levels of TNF-α, IL-1, IL-6, IL-10, IL-18 and othercytokines either by elisa or bioassay. Additional studies to evaluatethe effects of the synthetic compound of Formulae (I), (II), (IIA) and(IIB) on specific cytokine signaling pathways such as Caspase-activity(Nr-1, Nr.3), NF-kB, MAP-kinase activity (P38, ERK and JNK) will also beperformed.

Results

Preclinical studies demonstrated that murine RAW 264.7 cells treatedwith increasing doses of the synthetic compounds of Formulae (I) and(IIB), specifically those designated TTL1 and TTL3 herein, atconcentrations as high as 10 ug/ml showed similar viability compared tountreated controls indicating that the inhibitory effects of thesynthetic compounds of Formulae (I) and (IIB) on TNF-α synthesis werenot mediated by a direct cytotoxic effect.

Subsequent studies with the compound of Formula (I) as synthesizedaccording to Example 1, TTL1 (as synthesized according to Example 2) andTTL3 (as synthesized according to Example 4) demonstrated that TTL1exhibited approximately 10 fold greater activity compared to thecompound of Formula (I) as synthesized according to Example 1 ininhibiting TNF-α and IL-1 synthesis. TTL3, as synthesized according toExample 4, contains an additional chemical modification exhibitedapproximately 100 times greater activity than TTL1, as synthesizedaccording to Example 2. It is noted that similar to the compound ofFormula (I) as synthesized according to Example 1, neither analog TTL1nor TTL3 significantly inhibited IL-6 synthesis. TTL1 exhibited a ten(10)-fold greater activity compared to the compound of Formula (I) assynthesized according to Example 1 in inhibiting TNF-α and IL-1synthesis.

TTL3 which contains an additional chemical modification exhibitedapproximately 100 times greater activity than TTL1. It is againimportant to note that similar to the compound of Formula (I) assynthesized according to Example 1, neither analog significantlyinhibited IL-6 synthesis. TABLE 1 Inhibition of LPS-Induced TNF-αSynthesis by the Compound of Formula (I) and TTL1 Formula (I) Formula(I) Formula (I) TTL1 TTL1 TTL1 LPS (0.1 μg/ml) (1 μg/ml) (10 μg/ml) (0.1μg/ml) (1 μg/ml) (5.4 μg/ml) TNF-α (ng/ml) 120 108 67 50 57 60 38

TABLE 2 Inhibition of SAC-Induced TNF-α Synthesis by the Compound ofFormula (I) and TTL1 Formula (I) Formula (I) Formula (I) TTL1 TTL1 TTL1SAC (0.1 μg/ml) (1 μg/ml) (10 μg/ml) (0.1 μg/ml) (1 μg/ml) (5.4 μg/ml)TNF-α (ng/ml) 385 410 275 165 250 285 150

TABLE 3 Inhibition of SAC-Induced IL-1 Synthesis by the Compound ofFormula (I) and TTL1 Formula (I) Formula (I) Formula (I) TTL1 TTL1 TTL1SAC (0.1 μg/ml) (1 μg/ml) (10 μg/ml) (0.1 μg/ml) (1 μg/ml) (5.4 μg/ml)IL-1α (pg/ml) 700 1350 1050 350 950 400 300

TABLE 4 The Compound of Formula (I) and TTL1 Do Not Inhibit SAC-InducedIL-6 Synthesis Formula (I) Formula (I) Formula (I) TTL1 TTL1 TTL1 SAC(0.1 μg/ml) (1 μg/ml) (10 μg/ml) (0.1 μg/ml) (1 μg/ml) (5.4 μg/ml) IL-6(ng/ml) 75 65 90 80 83 86 65

TABLE 5 TTL3 Inhibits SAC-Induced TNF-α Synthesis 0.001 0.01 0.1Unstimulated SAC μg/ml μg/ml μg/ml 1 μg/ml 10 μg/ml TNF-α 5 375 80 75 8560 80 (ng/ml)

TABLE 6 TTL3 Inhibits SAC-Induced IL-1 Synthesis 0.001 0.01 0.1Unstimulated SAC μg/ml μg/ml μg/ml 1 μg/ml 10 μg/ml IL-1α 0 650 200 220190 180 170 (pg/ml)

TABLE 7 Inhibition of LPS-Induced TNF-α Synthesis by TTL3 (TNF-α(ng/ml)) LPS (1 μg/ml) + TTL3 (μg/ml) LPS alone (1 × 10⁻⁷) (1 × 10⁻⁶) (1× 10⁻⁵) (1 × 10⁻⁴) (1.0) (1 × 10²) 88 41 18 10 15 13 4

TABLE 8 TTL3 Inhibits Mortality After LPS/D-Gal Administration MortalityMortality Treatment* 24 hours 48 hours LPS/D-Gal 10/10  10/10 LPS/D-Gal + DMSO 8/10 9/10 LPS/D-Gal + TTL3 2/10 2/10*All treatments were i.p., TTL3 administration 45 minutes prior to LPS

While specific embodiments of the invention have been shown anddescribed in detail and exemplified to illustrate the application of andthe principles of the invention, it will be understood that theinvention may be embodied otherwise without departing from suchprinciples.

1. A method of treating a disease condition selected from the groupconsisting of inflammation, tuberculous pleurisy, rheumatoid pleurisy,cancer, cardiovascular disease, skin redness, diabetes, transplantrejection, otitis media (inner ear infection), sinusitis, and viralinfection comprising: identifying an animal with said disease condition;and contacting a compound to living tissue of said animal, wherein thecompound is:

wherein: if any R₃-R₅, R₇, R₈, R₁₁-R₁₅ is not hydrogen, R₂ or R₆ or R₉is not methyl, or R₁₀ is not CH₂, then R₁ is selected from the groupconsisting of hydrogen, a halogen, COOH, C₁-C₁₂ carboxylic acids, C₁-C₁₂acyl halides, C₁-C₁₂ acyl residues, C₁-C₁₂ esters, C₁-C₁₂ secondaryamides, (C₁-C₁₂)(C₁-C₁₂) tertiary amides, C₁-C₁₂ alcohols,(C₁-C₁₂)(C₁-C₁₂) ethers, C₁-C₁₂ alkyls, C₁-C₁₂ substituted alkyls,C₂-C₁₂ alkenyls, C₂-C₁₂ substituted alkenyls, and C₅-C₁₂ aryls; but ifall R₃-R₅, R₇, R₈, R₁₁-R₁₃ are hydrogen, R₂, R₆ and R₉ are each methyl,and R₁₀ is CH₂, then R₁ is selected from hydrogen, a halogen, C₁-C₁₂carboxylic acids, C₁-C₁₂ acyl halides, C₁-C₁₂ acyl residues, C₂-C₁₂esters, C₂-C₁₂ secondary amides, (C₁-C₁₂)(C₁-C₁₂) tertiary amides,C₂-C₁₂ alcohols, (C₁-C₁₂)(C₁-C₁₂) ethers other than methyl-acetyl ether,C₂-C₁₂ alkyls, C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyls, C₂-C₁₂substituted alkenyls, and C₂-C₁₂ aryls; R₂ and R₉ are each separatelyselected from hydrogen, a halogen, C₁-C₁₂ alkyl, C₁-C₁₂ substitutedalkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substituted alkenyl, C₂-C₁₂ alkynyl,C₁-C₁₂ alcohol, C₁-C₁₂ acyl, and C₅-C₁₂ aryl; R₃-R₅, R₇, R₈, and R₁₁-R₁₃are each separately selected from hydrogen, a halogen, C₁-C₁₂ alkyl,C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂ substituted alkenyl,C₂-C₁₂ alkynyl, and C₅-C₁₂ aryl; R₆ is selected from hydrogen, ahalogen, C₁-C₁₂ alkyl, C₁-C₁₂ substituted alkyls, C₂-C₁₂ alkenyl, C₂-C₁₂substituted alkenyl, and C₂-C₁₂ alkynyl; R₁₀ is selected from hydrogen,a halogen, CH₂, C₁-C₆ alkyl, C₁-C₆ substituted alkyl, C₂-C₆ alkenyl,C₂-C₆ substituted alkenyl, C₁-C₁₂ alcohol, and C₅-C₁₂ aryl; and R₁₄ andR₁₅ are separately selected from hydrogen, a halogen, CH₂, C₁-C₆ alkyl,C₁-C₆ substituted alkyl, C₂-C₆ alkenyl, C₂-C₆ substituted alkenyl, C₁-C₆alcohol, and C₅-C₆ aryl; wherein the compound includes the prodrugesters of the above compounds, and the acid-addition salts thereof. 2.The method of claim 1, wherein R₁ is selected from hydrogen, a halogen,C1 C12 carboxylic acids, C1 C12 acyl halides, C1 C12 acyl residues, C2C12 esters, C2 C12 secondary amides, (C1 C12)(C1 C12) tertiary amides,C2 C12 alcohols, (C1 C12)(C1 C12) ethers other than methyl-acetyl ether,C2 C12 alkyls, C1 C12 substituted alkyls, C2 C12 alkenyls, C2 C12substituted alkenyls, and C2 C12 aryls.
 3. The method of claim 1,wherein R₁ is selected from the group consisting of hydrogen, a halogen,COOH, C1 C12 carboxylic acids, C1 C12 acyl halides, C1 C12 acylresidues, C1 C12 esters, C1 C12 secondary amides, (C1 C12)(C1 C12)tertiary amides, C1 C12 alcohols, (C1 C12)(C1 C12) ethers, C1 C12alkyls, C1 C12 substituted alkyls, C2 C12 alkenyls, C2 C12 substitutedalkenyls, and C5 C12 aryls.
 4. The method of claim 1, wherein R₁ isselected from the group consisting of C₂-C₁₂ esters and C₁-C₁₂ acylresidues.
 5. The method of claim 1, wherein R₁ is selected from thegroup consisting of C₂-C₆ esters.
 6. The method of claim 1, wherein R₁₀is selected from the group consisting of C₂-C₆ alkyl groups and C₂-C₆alkenyl groups.
 7. The method of claim 1, wherein R₃-R₅, R₇, R₈, R₁₁-R₁₅is each hydrogen.
 8. The method of claim 1, wherein R₃-R₅, R₇, R₈,R₁₁-R₁₅ is each hydrogen; R₂, R₆, and R₉ are each methyl; and R₁₀ isCH₂.
 9. The method of claim 1, wherein R₁₅ is hydrogen, and R₁₄ isselected from hydrogen, a halogen, C₂-C₆ alcohols, C₂-C₆ alkyls, C₁-C₆substituted alkyls, C₂-C₆ alkenyls, C₂-C₆ substituted alkenyls, andC₅-C₆ aryls.
 10. A method of treating a disease condition selected fromthe group consisting of tuberculous pleurisy, rheumatoid pleurisy,cancer, cardiovascular disease, skin redness, diabetes, transplantrejection, otitis media (inner ear infection), sinusitis, and viralinfection comprising: identifying an animal with said disease condition;and contacting a compound selected from (a) acanthoic acid, (b)(−)-pimara-9(11), 15-dien-19-ol, (c) (−)-pimara-9(11), 15-dien-19-oicacid, (d) (−)-pimara-9(11), 15-dien-19-ol 19-acetate, (e)(−)-pimara-9(11), 15-diene, and (f) the methyl ester analog of acanthoicacid, to living tissue of said animal.